17 results on '"Magdalena Winiarska"'
Search Results
2. CD180 As a Putative Target for Immunotherapy in Aggressive Mature B Cell Lymphomas
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Klaudyna Fidyt, Marta Krawczyk, Monika Pepek, Katsiaryna Marhelava, Agnieszka Kraft, Malgorzata Bobrowicz, Aleksandra Kusowska, Joanna Barankiewicz, Przemyslaw Juszczynski, Michal Smida, Myron Czuczman, Francisco Hernandez-Ilizaliturri, Malgorzata Firczuk, and Magdalena Winiarska
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
3. Pharmacological Induction of NOXA Sensitizes High-Risk B Cell Acute Lymphoblastic Leukemia Cells to Venetoclax
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Angelika Muchowicz, Nicholas T. Crump, Julia Cyran, Malgorzata Firczuk, Karolina Siudakowska, Thomas A. Milne, Elżbieta Patkowska, Klaudyna Fidyt, Magdalena Winiarska, Agata Pastorczak, Lukasz Komorowski, Krzysztof Domka, Jakub Golab, Agnieszka Goral, and Agnieszka Graczyk-Jarzynka
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Stromal cell ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,chemistry.chemical_compound ,Leukemia ,chemistry ,Apoptosis ,Cell culture ,Cancer research ,medicine ,Cytotoxic T cell ,MTT assay ,Propidium iodide ,Thioredoxin - Abstract
Background: Venetoclax (VEN), a specific BCL2 inhibitor, exerts anti-leukemic effects in various high-risk (HR) B-ALL subtypes, such as ALL with mixed lineage leukemia (MLL) gene rearrangements (MLLr ALL) (PMID: 26711339), Philadelphia chromosome-positive (Ph+) (PMID 30546081) or hypodiploid B-ALL (PMID 30862722). Nevertheless, despite high rationale for targeting BCL2 in these subtypes of B-ALL, VEN monotherapy is not effective enough to completely eliminate leukemic cells. For this reason identification of other drugs that could sensitize leukemic cells to VEN may become beneficial treatment strategy in HR ALL. Previously, we showed that the enzymes of the thioredoxin system are upregulated in primary B-ALL cells and that auranofin (AUR), a thioredoxin reductase inhibitor, effectively kills leukemic cells in vitro and in vivo. Importantly, elements of the thioredoxin system are not only balancing redox homeostasis within the cells, but may also interact with other pathways, including anti-apoptotic signaling. Considering above, we hypothesized that AUR may potentiate VEN efficacy in HR B-ALL. Methods: To evaluate cytostatic/cytotoxic effects of VEN+AUR combination by MTT assay and propidium iodide (PI)-staining we used HR B-ALL cell lines, including SEM (MLLr ALL), BV-173 (Ph+ ALL) and NALM-16 (hypodiploid ALL). Patient derived xenograft cells (PDX) were generated through long-term propagation of primary B-ALL samples in immune-deficient NSG mice. Ex vivo drug testing in co-culture system was performed using primary bone marrow-derived mesenchymal stem cells (BM-MSC) and murine stromal OP9 cell line. NOXA genomic knockout (KO) in SEM cells was established by CRISPR/Cas9 system. Chromatin accessibility within PMAIP1 gene (encodes for NOXA) was detected using ATAC-seq. Results: We observed that AUR sensitizes HR B-ALL cell lines to VEN, as determined by MTT and PI-staining. Further, we mimicked the bone marrow support of stromal cells towards B-ALL and evaluated its impact on the response to VEN+AUR. For this reason we employed an ex vivo co-culture system of B-ALL PDX cells with primary BM-MSC or an OP9 cell line. In all tested PDX samples representing diagnostic/relapsed MLLr ALL (n=8), Ph+ ALL (n=2) and Ph-like ALL (n=2) we observed synergistic effect of this combination (Fig. 1A). Next, we determined the efficacy of VEN+AUR combination in vivo using a PDX model of MLLr B-ALL. We observed that administration of VEN+AUR diminished the progression of leukemia during a 3 week-long treatment more effectively than any single drug alone, which reflected in longer survival of NSG mice (Fig. 1B). Subsequently, we aimed to uncover the mechanism responsible for the synergistic action of VEN+AUR. In cells treated with both drugs we observed enhanced caspase activation and changes in the levels of BCL2 family proteins involved in apoptotic signaling. In particular, we found that AUR strongly upregulates a pro-apoptotic NOXA protein, both in HR B-ALL cell lines and in MLLr ALL PDX samples (Fig. 1C). To evaluate whether NOXA induction is functionally relevant for the cell death mediated by VEN+AUR, we generated SEM cells with a NOXA genomic KO. Lack of NOXA significantly abolished VEN-single agent as well as VEN+AUR combination cytotoxicity, demonstrating its dependence on NOXA expression (Fig. 1D). We then showed that NOXA is regulated at the transcriptional level, as co-treatment with AUR and the transcription inhibitor, actinomycin D, abolished AUR-mediated NOXA induction at mRNA and protein levels in SEM cells. Additionally, to test whether AUR-treatment itself provokes changes in chromatin accessibility within the NOXA encoding gene (PMAIP1) we performed ATAC-seq. We observed a clear increase in accessibility at PMAIP1 in response to AUR, which correlated with transcriptional induction of NOXA. Moreover, ChIP-qPCR revealed that increased ATAC peaks within PMAIP1 were associated with an increase in H3 lysine 27 acetylation (H3K27ac) - an epigenetic mark associated with open chromatin conformation. Conclusions: Our results demonstrate that FDA-approved drug, AUR, is a promising candidate to be used in combination with VEN for the therapy of HR B-ALL subtypes. Importantly, NOXA induction by AUR plays a central role in the VEN+AUR synergistic cytotoxicity. More studies elucidating the mechanism of NOXA upregulation by AUR are underway. Disclosures Milne: OxStem Oncology (OSO), a subsidiary company of OxStem Ltd.: Other: Founding shareholder .
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- 2020
4. Innate-like Chemokine Receptor Profile and Migratory Behaviour By Terminally Differentiated and Educated NK Cells
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Marianna Vincenti, Dennis Clement, Eivind Heggernes Ask, Merete Thune Wiiger, Hanna Julie Hoel, Magdalena Winiarska, Daniel Alfredo Palacios, Karl-Johan Malmberg, Radoslaw Zagozdzon, and Mieszko Lachota
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CCR1 ,CCR2 ,Chemokine ,T cell ,Immunology ,C-C chemokine receptor type 7 ,Cell Biology ,Hematology ,Biology ,CXCR3 ,Biochemistry ,Cell biology ,Chemokine receptor ,medicine.anatomical_structure ,medicine ,biology.protein ,CXC chemokine receptors - Abstract
Natural Killer (NK) cells play an essential role in cancer surveillance and have a unique capability of spontaneous cytotoxicity against cancer cells. The human NK cell repertoire is functionally diversified through a tightly regulated differentiation process characterized by an early transition from CD56bright to CD56dim NK cells, followed by coordinated changes in expression of inhibitory receptors, including NKG2A and killer cell immunoglobulin-like receptors (KIR). The acquisition of self HLA class I binding KIRs during NK cell differentiation tunes the cytotoxic potential of NK cells in a process termed education, characterized by increased loading of granzyme B in dense core granules. Although NK cell differentiation and education are critical determinants of the functional potential of the cell, little is known about how these events shape the migratory behavior of NK cells. To mediate appropriate and directed immune response against cancer, NK cells must be capable of migration to the tumor site. This process is mediated by chemokines, which guide cell migration by binding to their specific receptors. For example, in multiple myeloma, CXCR3 and CCR5 ligands (MIG, IP-10, and MIP-1a) are significantly upregulated in the bone marrow compared to healthy controls, affecting the composition of immune cells in the tumor microenvironment. In order to delineate the homing patterns of distinct NK cell subsets, we used high-dimensional flow cytometry combined with functional assays to map the NK cell chemokine receptor expression and migratory behavior. We screened resting and cytokine/feeder cell stimulated peripheral blood NK cells for the expression of a panel of 20 chemokine receptors (A). Based on CD56, CD57, NKG2A, and KIR expression, NK cells were divided into 6 phenotypically and functionally distinct subsets that were ordered according to their differentiation status (B). We found that the expression of CX3CR1, CXCR1, CXCR2, and CMKLR1 gradually increased during differentiation, whereas the expression of CXCR3, CCR7, and CCR5 was lower in more differentiated NK cells. CXCR4, CCR4, and CCR2 expression was relatively uniform across all subsets. Interestingly, CCR1 and CXCR6 were expressed mainly on less differentiated NKG2A+ CD56dim NK cells (B). Next, we stratified the chemokine receptor expression on mature KIR+ NK cells based on the expression of self (educated) or non-self KIR (uneducated). Educated NK cells expressed CXCR1, CX3CR1, CCR5, and CMKLR1 at higher levels than the uneducated NK cells. Conversely, CXCR3 was expressed at lower levels on educated NK cells (C). No difference was observed for CXCR2 expression. To determine whether the observed differences in chemokine receptor expression translate into altered chemokine responsiveness between the subsets, we combined the transwell system with multicolor flow cytometry. We found that the chemokine-induced migration capability of NK cells correlated closely with the expression level of corresponding chemokine receptor, leading to subset specific responses to various chemokine gradients (D). The present results show that peripheral blood NK cell chemokine receptor profile changes in a coordinated fashion during NK cell differentiation and is further influenced by the expression of self-specific KIR. Interestingly, receptors which expression declines during NK cell differentiation (CCR5, CCR7, and CXCR3) are commonly associated with adaptive T cell responses to viruses, whereas receptors that are upregulated along the differentiation axis (CXCR1, CXCR2, CX3CR1, CMKLR1) are typical for neutrophils and macrophages as a part of the innate immune response. Thus, our results suggest that NK cell differentiation and education processes together shape the NK cell migratory capabilities to promote homing of the most functional NK cell subsets to the site of inflammation and serve as the first line of defense in the immune response to pathogens and tumors. Figure Disclosures Malmberg: Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.
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- 2020
5. HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies
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Mikolaj Slabicki, Magdalena Gabrysiak, Elise Berthel, Mieszko Kozikowski, Nina Miazek, Beata Pyrzynska, Marta Karp, Dimitar G. Efremov, Agnieszka Graczyk-Jarzynka, Joanna Stachura, Luca Laurenti, Thorsten Zenz, Magdalena Winiarska, Antoni Domagala, Krzysztof Giannopoulos, Lukas P. Frenzel, Jakub Golab, Malgorzata Bobrowicz, Kamil Bojarczuk, Marta Siernicka, Ewa Lech-Marańda, Agata Malenda, Agata Malinowska, Dunja Baatout, Piotr Zapała, Jean-Jacques Diaz, Abdessamad Zerrouqi, Agnieszka Zagozdzon, Nicole Dalla-Venezia, Malgorzata Firczuk, Angelika Muchowicz, Joanna Barankiewicz, and Michal Dwojak
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0301 basic medicine ,Lymphoma ,Messenger ,Mice, SCID ,Pharmacology ,Histone Deacetylase 6 ,Biochemistry ,Mice ,immune system diseases ,hemic and lymphatic diseases ,Tumor Cells, Cultured ,Chronic ,Inbred BALB C ,Regulation of gene expression ,CD20 ,B-Lymphocytes ,Mice, Inbred BALB C ,Tumor ,Leukemia ,Cultured ,Lymphoma, Non-Hodgkin ,Hematology ,Lymphocytic ,Tumor Cells ,Up-Regulation ,3. Good health ,Gene Expression Regulation, Neoplastic ,Female ,Rituximab ,medicine.drug_class ,Immunology ,Non-Hodgkin ,Antineoplastic Agents ,Biology ,SCID ,Monoclonal antibody ,Histone Deacetylases ,Cell Line ,03 medical and health sciences ,Downregulation and upregulation ,Cell Line, Tumor ,medicine ,Animals ,Humans ,RNA, Messenger ,Epigenetics ,Antigens, CD20 ,Histone Deacetylase Inhibitors ,Leukemia, Lymphocytic, Chronic, B-Cell ,Antigens ,Neoplastic ,B-Cell ,Cell Biology ,HDAC6 ,Settore MED/15 - MALATTIE DEL SANGUE ,030104 developmental biology ,Gene Expression Regulation ,Cell culture ,biology.protein ,RNA ,Histone deacetylase - Abstract
Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.
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- 2017
6. SHP1 Deficiency Is Responsible for the Constitutive Activation of the BCR Pathway in GCB DLBCL
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Fabiola Porro, Engin Bojnik, Francesco Bertoni, Binu K. Sasi, Beata Pyrzynska, Abdessamad Zerrouqi, Richard Rosenquist, Dimitar G. Efremov, Magdalena Winiarska, Malgorzata Bobrowicz, Sven Turkalj, Valdemar Priebe, Hilal Kalkan, and Larry Mansouri
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medicine.medical_specialty ,Hematology ,Protein Tyrosine Phosphatase SHP-1 ,Venetoclax ,Btk inhibitors ,Immunology ,breakpoint cluster region ,Cell Biology ,NFKB1 ,medicine.disease ,Biochemistry ,chemistry.chemical_compound ,chemistry ,hemic and lymphatic diseases ,Internal medicine ,Ibrutinib ,medicine ,Cancer research ,Diffuse large B-cell lymphoma - Abstract
Functional and transcriptional profiling studies have identified a subset of diffuse large B cell lymphoma (DLBCL) tumors that rely on B cell receptor (BCR) signals for proliferation and survival. These BCR-dependent tumors have been identified among both the ABC and GCB DLBCL subtype. However, differences have been observed between the two subtypes in terms of the mechanism of BCR pathway activation and activity of downstream signaling molecules. BCR-dependent ABC DLBCL tumors are characterized by high basal NF-kB and PI3K/AKT activity and the presence of gain-of-function mutations in the CD79A or CD79B subunit of the BCR that are responsible for the chronic activation of the BCR pathway. In contrast, BCR-dependent GCB DLBCL tumors have low baseline NF-kB activity and lack CD79A/CD79B mutations, suggesting a different mechanism of BCR pathway activation. In this study, we investigated whether activation of the BCR pathway in GCB DLBCL is potentially caused by deficiency of the phosphatase SHP1, which is an important negative regulator of the BCR pathway and has been reported to be downregulated in approximately 40% of primary DLBCL tumors. For this purpose, we first correlated SHP1 expression with the presence of phosphorylated SYK and BLNK in the GCB DLBCL cell lines OCI-Ly1, OCI-Ly7, OCI-Ly18, SU-DHL-4, SU-DHL-6, WSU-NHL, SU-DHL-8, Toledo, BJAB, OCI-Ly19, OCI-Ly4, and Farage. Immunoblotting analysis revealed that SHP1 is expressed in SU-DHL-8, Toledo, BJAB, OCI-Ly19, OCI-Ly4, and Farage but not in the remaining 6 cell lines. Expression of SHP1 inversely correlated with expression of phosphorylated SYK (pSYK-Y352) and BLNK (pBLNK-Y84), suggesting that these two BCR-proximal signaling molecules are activated because of SHP1 deficiency. To further evaluate this possibility, we re-expressed SHP1 in OCI-Ly1 and SU-DHL-4 cells by transient transfection with a plasmid or a lentiviral SHP1 expression vector. Both approaches resulted in a substantial reduction of pSYK-Y352 and pBLNK-Y84 levels, confirming that SHP1 deficiency leads to activation of the BCR pathway. To determine whether activation of the BCR pathway contributes to tumor cell survival, we investigated the effects of the SYK inhibitor R406 (active substance of the drug fostamatinib) on the viability of the 6 SHP1-positive and 6 SHP1-negative GCB DLBCL cell lines. At concentrations up to 2 μM, R406 displayed only minimal toxicity against each of the SHP1-positive and SHP1-negative GCB DLBCL cell lines ( Disclosures No relevant conflicts of interest to declare.
- Published
- 2018
7. Dasatinib Effect on NK Cells and Anti-Tumor Immunity
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Zofia Pilch, Agnieszka Graczyk-Jarzynka, Mieszko Lachota, Magdalena Winiarska, and Marta Siernicka
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Chemokine ,biology ,medicine.drug_class ,business.industry ,medicine.medical_treatment ,Immunology ,Inflammation ,Cell Biology ,Hematology ,medicine.disease ,Monoclonal antibody ,Biochemistry ,Dasatinib ,Leukemia ,Cytokine ,Immunity ,Cell culture ,hemic and lymphatic diseases ,medicine ,Cancer research ,biology.protein ,medicine.symptom ,business ,medicine.drug - Abstract
Introduction Dasatinib is a potent small molecule kinase inhibitor targeting BCR-ABL kinase - oncogenic driver in Philadelphia chromosome-positive (Ph+) cases of chronic myelogenous leukemia (CML). In addition to BCR-ABL kinase, it also targets a broad array of other kinases, affecting not only leukemia cells but also immune cells. Just one hour after dasatinib oral administration a rapid increase of NK, NKT, T and B cells is observed in peripheral blood. Dasatinib has been also shown to influence NK cell cytotoxicity, however, the results are discordant. Some groups observe potentiation of NK cell cytotoxic activity while others strong inhibitory effects. These inconsistencies may be explained by differences in in vitro protocols used to study this phenomenon. Aim Our study aims to investigate dasatinib influence on immune cells in whole blood assays resembling physiological conditions observed in patients. In particular, we want to investigate dasatinib effect on NK cell mobilization, degranulation, and anti-tumor immunity. In light of clinical and pre-clinical studies involving dasatinib and the importance of NK cells in cancer, it is crucial to establish the mechanisms and kinetics of dasatinib immunomodulatory activity. Methods Before and one hour after first dasatinib administration peripheral blood was collected from CML patients. Collected whole blood was directly added to the target K562 cell line. After co-incubation, erythrocytes were lysed, cells were stained with a panel of monoclonal antibodies and analyzed with flow cytometry. A relative increase in lymphocyte count was determined by Trucount Tubes (BD). Dasatinib effect on NK cells in vitro was studied with degranulation and cytotoxicity assays using NK cells isolated from healthy volunteers PBMCs. Dasatinib at clinically relevant concentrations (20-200mM) was used to assess its effect on NK cell degranulation, cytotoxicity, cytokine, and chemokine production with flow cytometry upon staining with anti-CD107a, TNF-α, IFN-γ and CCL-4 monoclonal antibodies. For in vivo experiments C57BL/6 mice were inoculated with EL4 tumor cell line stably expressing luciferase and human CD20. 3 days after tumor inoculation mice were treated with dasatinib or vehicle intraperitoneally (i.p.) in a dose of 30 mg/kg. To monitor tumor growth, mice were injected with luciferin and imaged using the IVIS system. Results In agreement with previous reports, we confirm NK, NKT, T and B cell count increase in peripheral blood after dasatinib administration. To evaluate how dasatinib influences NK cell cytokine and chemokine production we stimulated NK cells with K562 cell line. Production of major proinflammatory cytokines secreted by NK cells, TNF-α and IFN-γ, was inhibited by dasatinib treatment. Production of MIP-1β (CCL4), a chemokine secreted by NK cells attracting a broad spectrum of immune cells to inflammation sites, was also profoundly decreased. According to our findings, dasatinib presence during the cytotoxicity assay, in a dose-dependent manner, inhibits NK cell cytotoxicity. However, 24-hour dasatinib pretreatment increases their cytotoxic potential. To better mimic the physiological conditions we used whole blood degranulation assay which closely resembles patient settings, including dasatinib concentration. One hour after dasatinib intake we observed a potent inhibitory effect of dasatinib on NK cell degranulation. Additionally, we observed a shift in NK cell subpopulations - dasatinib present during degranulation assay decreases CD16⁻ NK cell number. Finally, we evaluated the influence of high-dose dasatinib treatment on tumor rejection in mice. Mice treated with dasatinib exhibit significantly increased tumor growth compared with vehicle-treated mice. Conclusions Using whole blood degranulation assay and in vitro degranulation and cytotoxicity assays we report that dasatinib effect on NK cell cytotoxicity is dose- and time-dependent. Our results indicate that dasatinib has a dual effect on NK cell degranulation and affects other NK cell functions including cytokine production and migration. Further studies are needed to evaluate the significance of these findings. Disclosures No relevant conflicts of interest to declare.
- Published
- 2018
8. Lysosomal Disruption Augments Obinutuzumab-Induced Direct Cell Death
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Malgorzata Bobrowicz, Piotr Mrowka, Beata Pyrzynska, Magdalena Winiarska, Michal Dwojak, Joanna Stachura, Marta Siernicka, Malgorzata Firczuk, and Antoni Domagala
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0301 basic medicine ,Programmed cell death ,NADPH oxidase ,biology ,Immunology ,Siramesine ,Cell Biology ,Hematology ,Biochemistry ,Cell biology ,Raji cell ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,0302 clinical medicine ,Cell killing ,chemistry ,Obinutuzumab ,030220 oncology & carcinogenesis ,biology.protein ,medicine ,Cancer research ,Annexin A5 ,Cytotoxicity ,medicine.drug - Abstract
Anti-CD20 mononclonal antibodies (mAbs) have a well-established role in the treatment of B-cell lymphoid malignancies. In addition to classic Fc-dependent mechanisms, including antibody-dependent and complement-mediated cytotoxicity, the so-called type II mAbs induce direct cell death. It has been shown that obinutuzumab, without Fc-crosslinking agents or effector cells, triggers non-apoptotic, lysosomal-dependent programmed cell death (PCD). The mechanism of PCD is characterized by actin reorganization, followed by permeabilization of the lysosomal membrane and subsequent generation of reactive oxygen species (ROS) through NADPH oxidase. Although, mechanisms of PCD are well-described, little is known about factors influencing sensitivity of malignant B-cells to obinutuzumab-mediated direct cell killing. Strategies to improve PCD could be potentially exploited to eliminate malignant cells, which are refractory to conventional immunotherapy. In this study, we aimed to investigate the influence of lysosomotropic agent, chloroquine, on the efficacy of obinutuzumab-mediated cytotoxicity. As PCD is dependent on lysosomal destabilization, we hypothesized that combination of obinutuzumab with lysosome-destabilizing agent would result in increased cell death. In our study, we used a Burkitt lymphoma Raji cell line that is widely employed as a model to assess the efficacy of obinutuzumab. Raji cells were incubated with obinutuzumab, alone or in combination with increasing concentrations of chloroquine, followed by annexin V/PI staining. Chloroquine, significantly increased direct cell death induced by obinutuzumab, without being toxic alone. Those observations were further corroborated by cell staining with other viability dies - TO-PRO-3 iodide and 7-AAD.The efficacy of the tested combination was completely abrogated by cytochalasin D - an inhibitor of actin polymerization and concanamycin A - inhibitor of vacuolar ATPases that activate acidic vacuoles including lysosomes, suggesting that chloroquine and obinutuzumab share a common mechanism of action. Since chloroquine has been reported to promote ROS generation, we analyzed the production of ROS with DCFDA staining. We observed that chloroquine potentiated the oxidative effect of obinutuzumab. Consistently, the effect of the combination was completely abrogated by a cell-permeable ROS scavenger - Tiron. As obinutuzumab has been shown to induce ROS production via activation of NADPH oxidase, we investigated the influence of NADPH oxidase inhibitor - diphenylene iodonium (DPI) on the combination's efficacy. DPI only partially reversed the effect of obinutuzumab, while strongly decreasing the effect of the combination. Altogether, we show for the first time that chloroquine sensitizes cell to lysosomal cell death induced by obinutuzumab. The results of our study provide a strong rationale for combining obinutuzumab with lysosomotropic agents. These findings may have particular importance given the fact that another lysomotropic agent - siramesine has recently been shown to induce selective cytotoxicity in chronic lymphocytic leukemia (CLL). Disclosures No relevant conflicts of interest to declare.
- Published
- 2016
9. HDAC Inhibitors As Potential New Agents Improving the Efficacy of Monoclonal Antibodies
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Marta Siernicka, Jakub Golab, Agata Malenda, Malgorzata Bobrowicz, Ewa Lech-Marańda, Michal Dwojak, Tomasz Stoklosa, Magdalena Winiarska, Kamil Bojarczuk, and Beata Pyrzynska
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CD20 ,biology ,Immunology ,Cell Biology ,Hematology ,Ofatumumab ,Biochemistry ,Romidepsin ,chemistry.chemical_compound ,Complement inhibitor ,Trichostatin A ,chemistry ,hemic and lymphatic diseases ,Panobinostat ,medicine ,Cancer research ,biology.protein ,Histone deacetylase ,Vorinostat ,medicine.drug - Abstract
HDAC (histone deacetylase) inhibitors belong to a novel class of antitumor agents widely explored in hematology. So far two of them - romidepsin and vorinostat have been registered in cutaneous T-cell lymphoma, while panobinostat awaits FDA decision concerning its registration in multiple myeloma. Several other agents are currently being tested in clinical trials in lymphoma and myeloma patients and preliminary results suggest that they may be most effective when used in combinations with other anti-cancer drugs. Reports from others (Sugimoto et al. Biochem Biophys Res Commun 2009 Shimizu et al. Leukemia 2010) and the results from our group show that HDAC inhibitors augment the efficacy of anti-CD20 monoclonal antibodies (mAbs) in complement-dependent cytotoxicity (CDC) by up-regulating CD20 level. In our previous reports we have shown that HDAC6 isoform is involved in the regulation of CD20 degradation and trafficking. Since the efficacy of mAbs depends not only on the level of target molecule but is also regulated by the expression of complement regulatory proteins (mCRPs) - CD46, CD55 and CD59, we sought to investigate the influence of HDAC inhibitors on the expression of those molecules. Moreover, given the fact that HDAC inhibitors have been reported to regulate NK cells activity, we investigated their influence on antibody-dependent cell-mediated cytotoxicity (ADCC) which is one of the mechanisms of action of mAbs in vivo. The purpose of this study was to thoroughly investigate the influence of HDAC inhibition on the level of surface molecules – CD20 and CD38 – targets already used and tested in clinical trials in immunotherapy of B-cell malignancies as well as the expression of mCRPs. Using non-selective pan-inhibitors as well as selective inhibitors of particular HDAC classes and isoforms we sought to show the inhibition of which HDAC enzyme is the most effective in potentiating the outcome of mAbs based anti-tumor therapies. Since the results of clinical trials show that the use of HDAC inhibitors has several non-specific adverse effects, we hypothesize that selective inhibition of particular isoforms may considerably diminish these toxicities. Using flow cytometry we performed experiments assessing the level of surface CD20 CD38 and mCRPs – CD46, CD55 and CD59. We used established lymphoma, chronic lymphocytic leukemia (CLL) and myeloma cell lines as well as primary cells isolated from the patients suffering from CLL. We observed that the use of HDAC pan-inhibitors – trichostatin A (TsA) and vorinostat (Fig.1A) and selective HDAC6 inhibitors – tubacin and ACY1215 (ricolinostat) (Fig.1B) considerably improves the outcome of anti-CD20 mAbs – rituximab and ofatumumab in CDC in several B-cell lymphoma cell lines and in CLL primary samples (Fig. 1C). In our experiments we show that selective inhibition of HDAC6 in Raji cells does not influence the expression of mCRPs (Fig. 1D). However, we observed that pre-incubation with tubacin considerably increases the expression of CD38 (Fig. 1 D). Therefore, in our further studies we aim at expanding our observations to primary samples from multiple myeloma patients, especially since novel anti-CD38 mAbs are currently intensively explored in the therapy of this malignancy. Moreover, using flow cytometry we determined the influence of HDAC inhibition on degranulation of NK cells isolated from healthy donors in ADCC in vitro assay with rituximab and ofatumumab. Contrary to the reports of others, we observed no negative regulation of NK cells activity and their ability to kill target tumor cells. Therefore, since HDAC inhibitors seem to influence mainly CDC, we investigated the influence of specific HDAC inhibitors on the level of mCRPs. Those results suggest that specific inhibition of HDAC3 (Fig. 1E) and HDAC1/2 (Fig. 1F) are effective therapeutic strategies as they considerably decreased the expression of complement inhibitors and concomitantly increased the level of CD20 antigen. However, further studies including silencing of particular HDAC isoforms with shRNA are needed to confirm these findings. Altogether, our results strongly suggest that HDAC inhibitors potentiate the outcome of mAbs used in hematological malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
- Published
- 2014
10. Influence of Btk Inhibitors on Antitumor Activity of Natural Killer Cells
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Michal Dwojak, Kamil Bojarczuk, Magdalena Winiarska, Malgorzata Bobrowicz, Marta Siernicka, Daniel Olive, and Beata Pyrzynska
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Janus kinase 3 ,Immunology ,Degranulation ,Cell Biology ,Hematology ,Biology ,NKG2D ,Biochemistry ,Interleukin 21 ,medicine.anatomical_structure ,medicine ,Cancer research ,biology.protein ,Interleukin 12 ,Bruton's tyrosine kinase ,IL-2 receptor ,B cell - Abstract
Small molecule Bruton's tyrosine kinase (Btk) inhibitors are extensively studied in preclinical investigations and clinical trials in the treatment of hematological malignancies derived from B-cells. Ibrutinib, due to its safety and efficiency, has been recently approved for the treatment of patients with Chronic Lymphocytic Leukemia (CLL) who received at least one prior therapy. Moreover, another Btk inhibitor AVL-292 is currently tested in clinical trials in patients with B Cell Non-Hodgkin's Lymphomas, CLL and Waldenstrom Macroglobulinemia. Despite a huge therapeutic potential of Btk inhibitors we have recently demonstrated that both natural killer (NK) cells cytotoxicity and degranulation are significantly impaired upon ibrutinib treatment (Bojarczuk et al., Leukemia 2014). Since NK cells are effectors of innate immune system capable to kill tumor cells directly and in the mechanism of antibody dependent cell-mediated cytotoxicity (ADCC), which constitutes one of the major mechanism of monoclonal antibodies widely used in hematooncology, in our ongoing studies we are focused to determine in details the influence of various Btk inhibitors on NK cells cytotoxicity, degranulation, cytokine secretion and expression of activatory/inhibitory receptors. All experiments were performed fully in vitro using human primary NK cells isolated from PBMC of healthy donors as well as NK92 and NK92.CD16 cell lines. To determine cytolytic activity of NK cells CFSE/PI flow cytometry assay was used. Cytokine secretion and NK cells degranulation, evaluated as the expression of CD107a on the surface of NK cells, were determined with flow cytometry upon incubation with target cells for 4 h. Phenotype of NK cells pre-incubated with tested compounds was determined with flow cytometry using antibodies conjugated with fluorochromes. The initial results of our studies show that various Btk inhibitors differentially regulate NK cells antitumor activity. Ibrutinib and AVL-292 which covalently bind to cysteines at the position 481 significantly inhibit NK cells cytotoxicity. Interestingly, we have observed that pre-incubation of NK cells with ibrutinib as well as AVL-292 results in down-regulation of CD25 and CD69. This effect was not observed upon treatment of NK cells with CGI-1746, a reversible Btk inhibitor which blocks phosphorylation of BTK in Y551 and Y223. Expression of NK cells activatory receptors such as NKp30, NK44, NKp46, NKG2D, DNAM-1 and CD16 remained unchanged. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
- Published
- 2014
11. HDAC6 Inhibition Increases CD20 Level and Improves The Efficacy Of Anti-CD20 Monoclonal Antibodies
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Jakub Golab, Malgorzata Bobrowicz, Magdalena Winiarska, Michal Dwojak, and Kamil Bojarczuk
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CD20 ,biology ,medicine.drug_class ,Chronic lymphocytic leukemia ,Immunology ,Cell Biology ,Hematology ,Ofatumumab ,medicine.disease ,Monoclonal antibody ,Biochemistry ,Molecular biology ,Blot ,chemistry.chemical_compound ,chemistry ,Antigen ,immune system diseases ,hemic and lymphatic diseases ,Cancer research ,biology.protein ,medicine ,Propidium iodide ,Antibody - Abstract
CD20, an integrate membrane protein expressed on the surface of normal and malignant B-cells is widely used as a molecular target for monoclonal antibodies (mAbs) in the therapy of non-Hodgkin’s lymphomas and chronic lymphocytic leukemia (CLL). Accumulating evidence indicates that CD20 can be modulated at several levels, both transcriptional and posttranscriptional and its up-regulation would result in increased efficacy of anti-CD20 mAbs. CD20 antigen has been reported to be regulated epigenetically e.g. by histone deacetylases (HDACs). The results of our preliminary experiments show that use of non-selective HDAC inhibitors as well as blocking the activity of a single HDAC isoform - HDAC6 leads to up-regulation of CD20 protein in B-cell lymphoma cell lines and increase of the efficacy of therapy with anti-CD20 mAbs. Since HDAC6 is engaged mainly in the acetylation of non-histone substrates and the observed up-regulation of CD20 molecule does not seem to rely on transcriptional mechanisms we postulate that HDAC6 is engaged in processes of CD20 trafficking or/and degradation. CD20 being a membrane bound protein is most probably undergoing endocytosis. However, this process and the role of HDAC6 in its regulation has not been explored so far. The aim of this study was to understand how the inhibition of HDAC6 activity influences CD20 level in normal and malignant B-cells. We wanted to determine the mechanisms underlying this phenomena. This study required use of B-cell lymphoma cell lines as well as lymphocytes infected with Epstein-Barr virus and normal B- lymphocytes. Several HDAC pan-inhibitors and HDAC6 inhibitors were tested. To assess the membrane level of CD20 antigen, FITC-anti-CD20 staining was performed followed by cytometric analysis. The influence of HDACi on total level of CD20 protein was assessed in Western blotting. The complement-dependent cytotoxicity (CDC) assay was performed using rituximab and ofatumumab as well as human serum as a source of complement. Cell cytotoxicity was assessed by propidium iodide staining followed by cytometric analysis. The influence of HDAC inhibition on the transcription of CD20 was examined by qRT-PCR using SyBR Green and hydrolysis probes. The activity of CD20 promoter after inhibition of HDAC was assessed in Dual Luciferase Assay. The colocalisation of CD20 with other proteins that may influence its trafficking/degradation was assessed using immunocytochemistry with specific antibodies and observed under confocal microscope. The results of our study strongly suggest that combining HDACi with anti-CD20 antibodies can be an effective therapeutic modality for patients suffering from B-cell malignancies. Extensive experiments aiming at determining what factors are engaged in the regulation of CD20 by HDAC6 are planned. Disclosures: No relevant conflicts of interest to declare.
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- 2013
12. Inhibitors Of B-Cell Receptor Molecules Affect Surface CD20 and Impair Antitumor Activity Of Anti-CD20 Monoclonal Antibodies
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Kamil Bojarczuk, Magdalena Winiarska, Malgorzata Bobrowicz, Michal Dwojak, Nina Miazek, Piotr Zapala, Dimitar G Efremov, and Jakub Golab
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CD20 ,medicine.drug_class ,Immunology ,B-cell receptor ,breakpoint cluster region ,Syk ,Cell Biology ,Hematology ,Biology ,Monoclonal antibody ,Biochemistry ,Molecular biology ,Raji cell ,hemic and lymphatic diseases ,medicine ,biology.protein ,Cancer research ,Protein kinase B ,PI3K/AKT/mTOR pathway - 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.
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- 2013
13. Statins Increase Antileukemic Potency of Imatinib Through the Inhibition of MDR/ABCB1 and BCRP/ABCG2 Drug Transporters Activity
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Tomasz Stoklosa, Paweł Włodarski, Joanna Niesiobedzka-Krezel, Marek Jakóbisiak, Eliza Glodkowska-Mrowka, Piotr Mrowka, Magdalena Winiarska, Grzegorz W. Basak, and Ilona Seferynska
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ABL ,biology ,Immunology ,Imatinib ,Cell Biology ,Hematology ,Pharmacology ,Biochemistry ,Imatinib mesylate ,hemic and lymphatic diseases ,medicine ,biology.protein ,Lovastatin ,Efflux ,neoplasms ,Tyrosine kinase ,medicine.drug ,K562 cells ,P-glycoprotein - Abstract
Abstract 2742 The treatment of chronic myeloid leukemia (CML) was revolutionized by the introduction of tyrosine kinase inhibitors (TKIs) to the clinical practice. However, there is still a significant group of patients who due to drug resistance do not fully benefit from the therapy. Among various reasons of drug resistance, the changes of intracellular concentration of TKIs caused by the activity of drug transporters may have profound effect on clinical activity of the drug. Intracellular concentration of imatinib is a result of: drug influx into the cell and drug efflux out of the cell. The latter is mediated by ATP-binding casette (ABC) transporters - MDR/ABCB1 and BCRP/ABCG2. According to the recent knowledge modulation of membrane cholesterol level may change the activity of ABC transporters by changing their conformation. Hence we decided to evaluate potential effect of statins, widely used hypocholesterolemic drugs, on antileukemic activity of imatinib in cell lines transformed with BCR/ABL oncoprotein and primary human CML CD34+ cells. In a set of experiments involving cytotoxic assays (i.e. clonogenic assay, XTT, trypan blue exclusion assay) we revealed that lovastatin, a member of statin family, is able to enhance imatinib cytotoxicity both in established cell lines (32Dcl3 BCR/ABL+, K562) as well as in primary CML CD34+ cells obtained from patients in various stages of the disease, including those clinically resistant to imatinib with no detectable ABL kinase domain mutations. In the same time, lovastatin alone and in combination with imatinib did not influence BCR/ABL negative cells (32Dcl3 wild type, HL-60, CD34+ cells obtained from healthy blood donors). As measured using specific, fluorescent-labeled substrates for ABC transporters (rhodamine-123 and Bodipy-FL-prazosin respectively), lovastatin significantly inhibits MDR/ABCB1- and BCRP/ABCG2-mediated efflux capacity. Similar effects were observed for other statins. The addition of cholesterol to the samples previously incubated with lovastatin completely reversed inhibitory activity of statins. To confirm these results we examined direct changes of intracellular concentration of imatinib itself using radiolabeled 14C-imatinib. In these settings, statins caused 2–3-fold increase in intracellular imatinib concentration in murine and human cell lines (32Dcl3 BCR/ABL+, K562) and also in primary CML CD34+ cells including clones resistant to imatinib (Figure 1). Statins did not influence initial concentration of imatinib what may suggest that their effects on imatinib concentration are not mediated by changes in OCT-1 activity. In addition, statins did not influence expression of MDR/ABCB1 and BCRP/ABCG2 drug transporters measured using qPCR nor ABC proteins levels measured using Wester blotting. Combination of imatinib and lovastatin induced cell cycle arrest and increased percentage of apoptotic leukemic cells measured using flow cytometric analysis after propidium iodide staining. Lovastatin also reduced imatinib-induced phosphorylation of the adaptor protein CrkL. In summary, we show that statins are able to increase therapeutic efficacy of imatinib through the inhibition of drug efflux mediated by MDR/ABCB1 and BCRP/ABCG2 transporters. Our results suggest that statin-mediated depletion of cholesterol in the cell may change conformational status of the efflux pumps, including MDR/ABCB1 and BCRP/ABCG2 pumps involved in the transport of imatinib, and in that way modulate intracellular concentration of the drug. The addition of statins may become attractive treatment modality for the selected group of patients in chronic phase and blast crisis.Fig. 1Statins increase intracellular concentration of imatinib in BCR/ABL-positive cells.Fig. 1. Statins increase intracellular concentration of imatinib in BCR/ABL-positive cells. CML-CP, CML-BP–CD34+ cells obtained from CML patient in chronic phase or blast crisis respectively; *p Disclosures: No relevant conflicts of interest to declare.
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- 2011
14. Src Family Tyrosine Kinases Are Involved in the Transcriptional Regulation of CD20 Levels
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Grzegorz W. Basak, Malgorzata Wanczyk, Jakub Golab, Tomasz Stoklosa, Magdalena Winiarska, Kamil Bojarczuk, Michal Dwojak, Dominika Nowis, and Jacek Bil
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Kinase ,Immunology ,Cell Biology ,Hematology ,Biology ,SRC Family Tyrosine Kinase ,Biochemistry ,Molecular biology ,Raji cell ,Dasatinib ,immune system diseases ,LYN ,hemic and lymphatic diseases ,Transcriptional regulation ,medicine ,Transcription factor ,Proto-oncogene tyrosine-protein kinase Src ,medicine.drug - Abstract
Abstract 1661 Introduction: Anti-CD20 monoclonal antibodies (mAbs) have considerably improved the outcomes of patients with B-cell malignancies and reveal promising therapeutic activity in some autoimmune diseases. They eliminate B cells by triggering indirect effector mechanisms of the immune system, namely complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Unfortunately, the resistance to anti-CD20 mAb-based first line therapies has been a considerable clinical problem. The mechanisms of this resistance are still poorly understood. In an elegant in vitro study by van Meerten et al. a direct positive correlation between rituximab antitumor activity and CD20 levels has been demonstrated. Although for many years CD20 has been described as a stable antigen, accumulating evidence indicates that CD20 can be modulated at several levels, both transcriptional and posttranscriptional. The processes that lead to CD20 downregulation could potentially impair antitumor activity of rituximab-based therapies and lead to rituximab resistance. Src family tyrosine kinases (SFTKs) including Lyn, Fyn and Lck have been already reported by Deans et al. to associate with CD20 localized to lipid rafts. They were shown to be activated during anti-CD20 mAb-mediated apoptosis upon clustering of rafts. However, to the best of our knowledge, the role of SFTKs in the regulation of CD20 expression has not been studied so far. Objectives: The aim of this study was to explore the molecular basis for Src family tyrosine kinases- dependent regulation of CD20 levels in lymphoma cells. Results: In the initial experiments performed using flow cytometry we observed a significantly reduced binding of anti-CD20 mAb to Raji cells incubated for 48h with various inhibitors of Src kinases (dasatinib, PP2, nilotynib, bosutinib, saracatinib) (Fig.1A-E). Dasatinib also impaired the binding of rituximab to Raji cells (Fig.1F). Decreased binding of anti-CD20 mAb upon dasatinib treatment was observed in three additional lymphoma cell lines and primary cells isolated from patients with chronic lymphocytic leukemia. All tested SFTKs inhibitors impaired rituximab-mediated CDC (R-CDC) over a dose range of rituximab concentrations (1–100 ug/ml) in all lymphoma cell lines. Interestingly, in Raji cells incubated for 48h with dasatinib we also observed a dose-dependent reduction of total CD20 protein levels, when assayed by Western blotting (Fig.2A). Moreover, a 48-h incubation with dasatinib significantly reduced the transcription of cd20 gene, as assessed with RT-PCR (Fig.2B). To further elucidate the mechanism of transcriptional regulation of CD20 we performed qRT-PCR studies. A strongly reduced transcription of cd20 gene was observed in Raji cells over a dose range of dasatinib (20–200 nM) after 24- (Fig.2C) and 48h- incubation (Fig.2D). Additionally, the CD20 promoter activity measured with reporter Firefly luciferase assay has been reduced as early as 1 hour after dasatinib treatment (Fig.2E). To elucidate in more detail binding of transcription factors to the promoter of cd20 gene, a chromatin immunoprecipitation assay was performed. Our early results indicate that dasatinib impairs binding of PU.1 transcription factor to its consensus site within cd20 promoter in Raji cells. Conclusions: Our studies indicate for the first time that SFTKs are involved in the transcriptional regulation of CD20 levels in lymphoma cells. Elucidation of the exact mechanism of this phenomena needs further studies. Results of these experiments will help to understand the biology and regulation of CD20 levels in lymphoma cells. The research was supported by Polish Ministry of Science and Higher Education [N N402 352938 (MW), IP2010/046570 (MW), IP2010/028670 (DN)]. Disclosures: No relevant conflicts of interest to declare.
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- 2011
15. Prenyl Transferases Are Involved in the Regulation of CD20 Levels and Influence Anti-CD20 Monoclonal Antibody-Mediated Activation of Complement-Dependent Cytotoxicity
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Dominika Nowis, Malgorzata Wachowska, Malgorzata Wanczyk, Tomasz Stoklosa, Magdalena Winiarska, Eliza Glodkowska-Mrowka, Jacek Bil, Michal Dwojak, Jakub Golab, Marta Miaczynska, Justyna Chlebowska, Kamil Bojarczuk, Ewa Wilczek, Angelika Muchowicz, and Malgorzata Firczuk
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Antibody-dependent cell-mediated cytotoxicity ,Geranylgeranyl Transferase ,biology ,Farnesyl Transferase Inhibitor ,Farnesyltransferase ,Immunology ,Cell Biology ,Hematology ,Ofatumumab ,Biochemistry ,Molecular biology ,Complement-dependent cytotoxicity ,Raji cell ,chemistry.chemical_compound ,Prenylation ,chemistry ,immune system diseases ,hemic and lymphatic diseases ,Cancer research ,biology.protein - Abstract
Abstract 3722 Anti-CD20 monoclonal antibodies (mAbs) (rituximab or ofatumumab) are being successfully used in the treatment of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). They exert antitumor effects by triggering indirect effector mechanisms of the immune system, such as activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, upon crosslinking with secondary antibodies, anti-CD20 mAbs can induce cell death. It is frequently underscored that CD20 expression levels in various B-cell tumors is relatively constant. However, accumulating evidence indicates that CD20 can be modulated at transcriptional, posttranscriptional, and even posttranslational levels. Moreover, it has been clearly shown in vitro that CDC efficacy of anti-CD20 mAbs clearly depends on CD20 expression. We have previously observed that statins impair detection of CD20 in non-Hodgkin lymphoma cells and impair rituximab-mediated CDC and ADCC (Winiarska et al. PLoS Med 2008). Statins are inhibitors of cholesterol synthesis and decrease production of prenyl groups (farnesyl and geranylgeranyl PPi), which are necessary for posttranslational modification of approximately 1% of cellular proteins. In experiments aimed at elucidation of the molecular mechanisms of statin-mediated modulation of CD20 we observed that neither geranylgeranyl transferase (GGT) nor farnesyl transferase (FNT) inhibitors could mimic statins effects. On the contrary, prenyltransferase inhibitors improved rituximab-mediated CDC. Therefore, we decided to investigate in more detail the interaction of prenyltransferase inhibitors and anti-CD20 mAbs. In the initial experiments we evaluated the effects of three different farnesyl transferase inhibitors as well as three different geranylgeranyl transferase inhibitors. Among all FNT and GGT inhibitors, L-744,832 seemed to produce the most significant influence on both rituximab-mediated CDC and CD20 levels (Figure). Moreover, L-744,832 significantly increased rituximab-mediated CDC in 3 out of 5 primary tumor cell cultures isolated from patients with NHL or CLL. Therefore, L-744,832 was selected for further more systematic studies. Interestingly, in Raji cells L-744,832 did not improve rituximab-mediated ADCC and only at the highest non-toxic concentrations it sensitized to rituximab+crosslinking antibody-mediated cytotoxicity. In 10 out of 17 (58.8%) primary lymphoma/leukemia cells L-744,832 increased CD20 expression by at least 20% as measured with flow cytometry. Moreover, we observed that upon L-744,832 treatment CD20 is up-regulated in Raji cells at both mRNA as well as protein level. Experiments aimed at investigation of FTI influence on proteasome activity as well as CD20 endocytosis and shedding revealed that L-744,832 influences CD20 levels independently from its posttranslational regulation. To verify whether modulation of CD20 levels by L-744,832 results from specific inhibition of farnesyltransferase or is an off-target effect of this compound we performed FNT B subunit (FNTB) knock-down experiments that resulted in increased CD20 levels by almost 60%. Incubation of Raji cells with a transcription inhibitor cycloheximide completely prevented L-744,832-mediated increase of CD20 levels in WB. Therefore, a chromatin immunoprecipitation assay was performed to determine whether inhibition of FNT activity is associated with binding of transcription factors to the promoter of cd20 gene. These studies revealed that L-744,832 promotes binding of PU.1 and Oct2, but not TFE3 to target DNA sequences within cd20 promoter in Raji cells. To conclude, our studies indicate for the first time that CD20 expression can be modulated by prenyltransferase inhibitors. While inhibition of FNT activity significantly up-regulates expression of CD20, the influence of GGT inhibitors on this protein is more complex, and requires further studies. Furthermore, pre-incubation of NHL and CLL cells with L-744,832, a FNT inhibitor, potentiates anti-CD20 mAb-mediated activation of the complement-mediated cytotoxicity. Therefore, the combination seems to be promising and its efficacy should be determined in patients with NHL or CLL. Disclosures: Winiarska: Genmab A/S: Research Funding. Golab:Genmab A/S: Research Funding.
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- 2011
16. Sorafenib Affects Membrane Complement Inhibitors and Improves Antitumor Activity of Rituximab
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Kamil Bojarczuk, Dominika Nowis, Marcin Okroj, Magdalena Winiarska, Grzegorz W. Basak, Jacek Bil, Jakub Golab, and Tomasz Stoklosa
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Sorafenib ,Antibody-dependent cell-mediated cytotoxicity ,CD20 ,biology ,business.industry ,medicine.drug_class ,Chronic lymphocytic leukemia ,Immunology ,Cell Biology ,Hematology ,Monoclonal antibody ,medicine.disease ,Biochemistry ,Antigen ,immune system diseases ,hemic and lymphatic diseases ,Cancer research ,biology.protein ,Medicine ,Rituximab ,Antibody ,business ,medicine.drug - Abstract
Abstract 3723 Monoclonal antibodies (mAbs) targeting CD20 antigen are now routinely used in the treatment of various types of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). Their antitumor action results from the ability to trigger complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, direct cell death can be induced upon crosslinking with secondary antibodies. Previous studies have demonstrated a sigmoidal correlation between CD20 expression level and rituximab-mediated CDC but not ADCC. Next to CD20 expression level also other potential mechanisms of anti-CD20-mediated cytotoxicity have been observed. Among them an increased expression of complement regulatory proteins (CRP), such as CD46, CD55 or CD59 has been shown to contribute to resistance to mAb-mediated CDC. Antibodies blocking CRP or siRNA that knocks-down CRP expression facilitate rituximab-mediated cytotoxicity. Also in xenograft in vivo models it was shown that antibodies blocking the activity of CD55 and CD59 or a recombinant adenoviral fiber knob protein that cross-links CD46 molecules enhance therapeutic effects of rituximab. Fludarabine, the nucleoside analogue clinically active against CLL and indolent NHL has been shown to act synergistically with rituximab and to down-regulate the membrane expression of CD55 without significantly altering CD20 levels. The aim of this study was to investigate the influence of sorafenib, a multikinase inhibitor on the expression of CRPs in human CD20+ B-cell lymphomas and to evaluate its ability to trigger CDC in combination with rituximab. A 48-h incubation of four different human non-Hodgkin lymphoma cells DoHH2 (Fig. 1A), Daudi, Raji and Ramos with sorafenib resulted in a marked concentration-dependent drop in the surface expression of CD46, CD55 and CD59, but did not affect the levels of CD20. Analysis of primary cells isolated from 5 consecutive patients with CD20+ CLL also revealed a significant drop in the levels of CD46 (5 of 5 patients), CD55 (4 of 5 patients), and CD55 (5 of 5 patients) after a 48 h incubation of tumor cells with sorafenib. Accordingly, with previous reports, sorafenib significantly decreased phosphorylation of STAT3 transcription factors that are involved in the regulation of CRP expression. Incubation of tumor cells with sorafenib lowered the levels of mRNA for all three CRPs, but not that of CD20 indicating that sorafenib-mediated downregulation of CRPs occurs at transcriptional level. Pre-incubation of all four lymphoma cell lines cells with sorafenib at concentrations that down-modulate CRPs levels also significantly sensitized tumor cells to rituximab-mediated CDC (Fig. 1B). Moreover, sorafenib led to increased incorporation of membrane-attack complex (C5b-9) into the membranes of DoHH2 cells. On the other hand, sorafenib did not affect rituximab-mediated ADCC nor apoptosis in DoHH2 cells. Sunitinib, another multi-kinase inhibitor, did not affect rituximab-mediated complement-dependent cytolysis.Figure 1.(A) DoHH2 cells were incubated with either diluent or sorafenib at 1.25, 2.5 and 3.75 μM concentration for 48h. Then, cells (1 × 105/100 μl) were stained for CD20, CD46, CD55 and CD59 and analyzed in a FACSCalibur using Cell-Quest Pro software version 5.2. (B) DoHH2, Daudi, Raji or Ramos cells were incubated with either diluent or sorafenib at appropriate concentrations for 48h. Then, equal numbers of cells (1 × 105/well) were incubated for 1h with serial dilutions (from 1 to 100 μg/ml) of rituximab in the presence of 10% human AB serum as a complement source. Cell viability was measured with a MTT assay. The survival of cells is presented as percentage of corresponding diluent- or sorafenib-pretreated cells without rituximab. *P Altogether, these results indicate that sorafenib, which undergoes clinical trials in patients with NHL and B-CLL might be effectively used in combination with anti-CD20 mAbs. Disclosures: No relevant conflicts of interest to declare.
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- 2011
17. Statins Impair Antitumor Effects of CD20 mAb by Inducing Conformational Changes of CD20
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Grzegorz M. Wilczynski, Eliza Glodkowska, Zbigniew Gaciong, Jakub Golab, Anna Dabrowska-Iwanicka, Tomasz Stoklosa, Małgorzata Lekka, Wendy J.M. Mackus, Tadeusz Issat, Jacek Bil, Maciej Siński, Elżbieta Górska, Patrick J. Engelberts, Krzysztof Warzocha, Marcin Makowski, Marek Jakóbisiak, Paul W. H. I. Parren, Piotr Mrowka, Dominika Nowis, Witold Lasek, M Wasik, Ewa Wilczek, and Magdalena Winiarska
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Statin ,medicine.drug_class ,medicine.medical_treatment ,Immunology ,Pharmacology ,Ofatumumab ,Monoclonal antibody ,Biochemistry ,chemistry.chemical_compound ,immune system diseases ,hemic and lymphatic diseases ,medicine ,Cytotoxicity ,B cell ,CD20 ,biology ,business.industry ,Cell Biology ,Hematology ,Immunotherapy ,medicine.anatomical_structure ,chemistry ,biology.protein ,Rituximab ,business ,medicine.drug - Abstract
A number of monoclonal antibodies (mAb) are presently in development or approved for CD20-directed immunotherapy. Ofatumumab, a human IgG1 anti-CD20 mAb, is currently being evaluated in phase III clinical trials for B-CLL and FL, and in phase II clinical trials for rheumatoid arthritis (RA). Rituximab, a chimeric IgG1 anti-CD20 mAb, has been approved for 1st line treatment of CD20-positive B cell lymphomas alone or in combination with chemotherapy, and in RA. Virtually all rituximab-treated patients relapse after single-agent treatment. The clinical efficacy of rituximab might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising anti-lymphoma effects. We studied the influence of statins on ofatumumab- and rituximab-mediated cytotoxicity. Surprisingly, B cells incubated with statins showed decreased rather than increased CD20 mAb-mediated complement-dependent cytotoxicity (CDC) in cell viability assays. However, cell lysis of statin-treated B cells remained higher when using ofatumumab in comparison to rituximab. Statins decreased CD20 immunostaining in flow cytometry but did not affect total cellular CD20 levels when compared to non-treated cells. Incubation of B cells with other cholesterol depleting agents (methyl-β-cyclodextrin (MβCD) and berberine) established that the presence of plasma membrane cholesterol and not lipid rafts may be required for CD20 mAb-mediated CDC. Cholesterol restitution reversed ofatumumab- and rituximab-mediated CDC and CD20 mAb staining. Statin incubation resulted in conformational changes of CD20 and impaired binding of CD20 mAb to the CD20 molecule as observed by atomic force microscopy. Freshly isolated cells of 4 B cell lymphoma patients were treated with the cholesterol-depleting agent MβCD, and CD20 mAb (B9E9) binding and rituximab-mediated CDC were significantly decreased (P
- Published
- 2007
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