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1.
Bruton’s tyrosine kinase (BTK) is a key regulator of B-cell receptor (BCR) signaling pathway and takes effect in the regulation of B-cell activation, survival, proliferation and differentiation. It has been proved that BTK is commonly overexpressed in mantle cell lymphoma (MCL), which makes it a focus of targeted therapy for MCL. Our studies yielded a novel series of pyrazolopyrimidine derivatives capable of potent inhibition of BTK. Notably, 12a showed higher selectivity against BTK and exhibited robust antiproliferative effects in both mantle cell lymphoma cell lines and primary patient tumor cells. Low micromolar doses of 12a induced strong cell apoptosis in Jeko-1 and Z138 cells.  相似文献   

2.
Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.  相似文献   

3.
The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK⿿s interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK⿿s binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK⿿s interaction with Importin 7.  相似文献   

4.
《Autophagy》2013,9(5):612-620
Glucocorticosteroid hormones, including prednisone and dexamethasone (Dex), have been used to treat lymphoid malignancies for many years because they readily induce apoptosis in immature lymphocytes lacking Bcl-2. However, elevated expression of the anti-apoptotic protein Bcl-2 inhibits apoptosis and contributes to glucocorticoid resistance. Using the Bcl-2-negative WEHI7.2 lymphoma line as an experimental model, we found that Dex not only induces apoptosis but also induces autophagy. The caspase inhibitor Z-VAD-fmk inhibited apoptosis but not autophagy in Dex-treated cells. Bcl-2 overexpression inhibited Dex-induced apoptosis even more potently than Z-VAD-fmk and, contrary to previous reports, Bcl-2 neither interacted with Beclin-1 nor inhibited autophagy. Rather, Bcl-2 overexpression facilitated detection of Dex-induced autophagy by both steady state methods and flux measurements, ostensibly due to apoptosis inhibition. Autophagy contributed to prolonged survival of Bcl-2-positive lymphoma cells following Dex treatment, as survival was reduced when autophagy was inhibited by 3-methyladenine. These findings emphasize the important interplay between apoptosis and autophagy and suggest a novel mechanism by which Bcl-2, which is frequently elevated in lymphoid malignancies, contributes to glucocorticoid resistance and survival of lymphoma cells.  相似文献   

5.
Glucocorticosteroid hormones, including prednisone and dexamethasone (Dex), have been used to treat lymphoid malignancies for many years because they readily induce apoptosis in immature lymphocytes lacking Bcl-2. However, elevated expression of the anti-apoptotic protein Bcl-2 inhibits apoptosis and contributes to glucocorticoid resistance. Using the Bcl-2-negative WEHI7.2 lymphoma line as an experimental model, we found that Dex not only induces apoptosis but also induces autophagy. The caspase inhibitor Z-VAD-fmk inhibited apoptosis but not autophagy in Dex-treated cells. Bcl-2 overexpression inhibited Dex-induced apoptosis even more potently than Z-VAD-fmk and, contrary to previous reports, Bcl-2 neither interacted with Beclin-1 nor inhibited autophagy. Rather, Bcl-2 overexpression facilitated detection of Dex-induced autophagy by both steady state methods and flux measurements, ostensibly due to apoptosis inhibition. Autophagy contributed to prolonged survival of Bcl-2-positive lymphoma cells following Dex treatment, as survival was reduced when autophagy was inhibited by 3-methyladenine. These findings emphasize the important interplay between apoptosis and autophagy and suggest a novel mechanism by which Bcl-2, which is frequently elevated in lymphoid malignancies, contributes to glucocorticoid resistance and survival of lymphoma cells.  相似文献   

6.
7.
Enzastaurin is an investigational PKCβ inhibitor that has growth inhibitory and pro-apoptotic effects in both B and T-cell lymphomas. We investigated the cytotoxicity and mechanisms of cell death of the combination of enzastaurin and low concentrations of histone deacetylase (HDAC) inhibitors in B-cell and T-cell lymphoma cell lines and primary lymphoma/leukemia cells. Combined enzastaurin/suberoylanilide hydroxamic acid treatment synergistically induced apoptosis in diffuse large B-cell lymphoma and T-cell lymphoma cell lines, and primary lymphoma/leukemia samples. Similarly, combined treatment of B-cell-like lymphoma cells with enzastaurin and two different HDAC inhibitors, valproic acid and (2E,4E)-6-(4-chlorophenylsulfanyl)-2,4-hexadienoic acid hydroxyamide synergistically induced apoptosis, suggesting the synergy is generalizable to other HDAC inhibitors. Our data indicate that enzastaurin/HDAC inhibitors therapy can synergistically inhibit growth and induce apoptosis in lymphoid malignancies and may be an effective therapeutic strategy. Potential mechanisms including enzastaurin mediated inhibition of HDAC inhibitor-induced compensatory survival pathways are discussed.  相似文献   

8.
BackgroundMyricetin (MYR) is a polyhydroxy flavone originally isolated from Myrica rubra, and is widely distributed in a variety of medicinal plants and delicious food. MYR has been proven to have inhibitory effects against various types of cancer. However, the exact role of MYR in lymphoma development is still unclear.MethodsIn vitro, the MTT assay was performed to evaluate the activity of human diffuse large B lymphoma cell TMD-8 and other tumor cells. Homogeneous time-resolved fluorescence (HTRF) and molecular docking were used to detect the target of MYR inhibiting TMD-8 cells. In addition, flow cytometry, Annexin V-FITC/PI assays, Hoechst 33258, and mondansylcadaverine (MDC) fluorescent standing were used to detect the cell cycle, apoptosis, and autophagy, respectively. Moreover, Western blot analysis was conducted to analyze related signaling pathways. In TMD-8 cell xenotransplanted mice, immunohistochemistry, histopathology, and blood biochemical tests were used to evaluate the effectiveness and safety of oral administration of MYR.ResultsHere, we found that MYR is more sensitive to TMD-8 cells than other tumor cells by targeting bruton tyrosine kinase (BTK). BTK is an attractive target for the treatment of B-cell malignancies. The HTRF assay showed that MYR inhibited BTK kinase with an IC50 of 1.82 μM. Furthermore, the HTRF assay and Western blot analysis demonstrated that MYR could bind to key residues (Ala478, Leu408, Thr474) in the BTK active pocket, inhibit the autophosphorylation on tyrosine 223, and block BTK/ERK and BTK/AKT signal transduction cascades (including downstream substrates GSK-3β, IKK, STAT3, and NF-κb). The results of cell cycle, apoptosis, and autophagy showed that MYR could induce G1/G0 cycle arrest by regulating cyclinB1/D1 expression, induce apoptosis by increasing the Bax/Bcl-2 ratio, and trigger autophagy by inhibiting mTOR activation. In vivo, oral administration of MYR significantly inhibited the growth of TMD-8 xenograft tumora without toxic side effects. Furthermore, Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis showed that MYR could inhibit proliferation and induce apoptosis of tissue lymphoma cells.ConclusionTaken together, MYR is an oral available natural BTK inhibitor that effectively inhibits the growth of lymphoma TMD-8 cells both in vitro and in vivo. In addition, our findings support that the use of MYR is a novel and promising therapeutic strategy for the treatment of lymphoma.  相似文献   

9.

Introduction

Spleen tyrosine kinase (SYK) is a key integrator of intracellular signals triggered by activated immunoreceptors, including Bcell receptors (BCR) and Fc receptors, which are important for the development and function of lymphoid cells. Given the clinical efficacy of Bcell depletion in the treatment of rheumatoid arthritis and multiple sclerosis, pharmacological modulation of Bcells using orally active small molecules that selectively target SYK presents an attractive alternative therapeutic strategy.

Methods

A SYK inhibitor was developed and assayed in various in vitro systems and in the mouse model of collagen-induced arthritis (mCIA).

Results

A novel ATP-competitive inhibitor of SYK, 6-[(1R,2S)-2-Amino-cyclohexylamino]-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylic acid amide, designated RO9021, with an adequate kinase selectivity profile and oral bioavailability, was developed. In addition to suppression of BCR signaling in human peripheral blood mononuclear cells (PBMC) and whole blood, FcγR signaling in human monocytes, and FcϵR signaling in human mast cells, RO9021 blocked osteoclastogenesis from mouse bone marrow macrophages in vitro. Interestingly, Toll-like Receptor (TLR) 9 signaling in human Bcells was inhibited by RO9021, resulting in decreased levels of plasmablasts, immunoglobulin (Ig) M and IgG upon B-cell differentiation. RO9021 also potently inhibited type I interferon production by human plasmacytoid dendritic cells (pDC) upon TLR9 activation. This effect is specific to TLR9 as RO9021 did not inhibit TLR4- or JAK-STAT-mediated signaling. Finally, oral administration of RO9021 inhibited arthritis progression in the mCIA model, with observable pharmacokinetics (PK)-pharmacodynamic (PD) correlation.

Conclusions

Inhibition of SYK kinase activity impinges on various innate and adaptive immune responses. RO9021 could serve as a starting point for the development of selective SYK inhibitors for the treatment of inflammation-related and autoimmune-related disorders.  相似文献   

10.
Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and secondary resistance to currently available therapies in most cases. Therefore, despite recent advances in the treatment of this disease, it is still considered to be incurable in the majority of cases. MCL B cells retain their B cell antigen receptor (BCR) expression during and after neoplastic transformation. BCRs in MCL show distinct patterns of antigen selection and ongoing BCR signaling. However, little is known about the involved antigens and the mechanisms leading to lymphomagenesis and lymphoma progression in MCL. Recent preclinical and clinical studies have established a crucial role of the BCR and the potential of inhibiting its signaling in this disease. This has established the B cell antigen receptor signaling cascade as a very promising therapeutic target to improve outcome in MCL alone or in combination with chemo-immunotherapy in recent years.  相似文献   

11.
A new family of diphenylpyrimidine derivatives bearing an amino acid substituent were identified as potent BTK inhibitors. Among them, compound 7b, which features an l-proline substituent, was identified as the strongest BTK inhibitor, with an IC50 of 8.7?nM. Compound 7b also displayed similar activity against B-cell lymphoma cell lines as ibrutinib. Moreover, 7b exhibited low cytotoxic activity against normal PBMC cells. In addition, the acridine orange/ethidium bromide (AO/EB) staining assay, Western blot analysis and flow cytometry analysis also showed its effectiveness in interfering with B-cell lymphoma cell growth. The molecular simulation performance showed that 7b forms additional strong hydrogen bonds with the BTK protein. All these findings provided new clues about the pyrimidine scaffold as an effective BTK inhibitor for the treatment of B-cell lymphoma.  相似文献   

12.
BackgroundBruton's tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) pathway and a clinically validated target for small molecule inhibitors such as ibrutinib in the treatment of B-cell malignancies. Tirabrutinib (GS-4059/ONO-4059) is a selective, once daily, oral BTK inhibitor with clinical activity against many relapsed/refractory B-cell malignancies.MethodsCovalent binding of tirabrutinib to BTK Cys-481 was assessed by LC-MSMS analysis of BTK using compound as a variable modification search parameter. Inhibition potency of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib against BTK and related kinases was studied in a dose-dependent manner either after a fixed incubation time (as used in conventional IC50 studies) or following a time course where inactivation kinetics were measured.ResultsTirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency kinact/Ki was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a kinact/Ki value of 2.4 ± 0.6 × 104 M−1 s−1 for BTK with selectivity against important off-targets.ConclusionsFor the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety.General significanceThis is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases.  相似文献   

13.
14.
15.
Aberrant microRNA (miR) expression plays an important role in pathogenesis of different types of cancers, including B-cell lymphoid malignancies and in the development of chemo-sensitivity or -resistance in chronic lymphocytic leukemia (CLL) as well as diffuse large B-cell lymphoma (DLBCL). Ibrutinib is a first-in class, oral, covalent Bruton’s tyrosine kinase (BTK) inhibitor (BTKi) that has shown impressive clinical activity, yet many ibrutinib-treated patients relapse or develop resistance over time. We have reported that acquired resistance to ibrutinib is associated with downregulation of tumor suppressor protein PTEN and activation of the PI3K/AKT pathway. Yet how PTEN mediates chemoresistance in B-cell malignancies is not clear. We now show that the BTKi ibrutinib and a second-generation compound, acalabrutinib downregulate miRNAs located in the 14q32 miRNA cluster region, including miR-494, miR-495, and miR-543. BTKi-resistant CLL and DLBCL cells had striking overexpression of miR-494, miR-495, miR-543, and reduced PTEN expression, indicating further regulation of the PI3K/AKT/mTOR pathway in acquired BTKi resistance. Additionally, unlike ibrutinib-sensitive CLL patient samples, those with resistance to ibrutinib treatment, demonstrated upregulation of 14q32 cluster miRNAs, including miR-494, miR-495, and miR-543 and decreased pten mRNA expression. Luciferase reporter gene assay showed that miR-494 directly targeted and suppressed PTEN expression by recognizing two conserved binding sites in the PTEN 3′-UTR, and subsequently activated AKTSer473. Importantly, overexpression of a miR-494 mimic abrogated both PTEN mRNA and protein levels, further indicating regulation of apoptosis by PTEN/AKT/mTOR. Conversely, overexpression of a miR-494 inhibitor in BTKi-resistant cells restored PTEN mRNA and protein levels, thereby sensitizing cells to BTKi-induced apoptosis. Inhibition of miR-494 and miR-495 sensitized cells by cooperative targeting of pten, with additional miRNAs in the 14q32 cluster that target pten able to contribute to its regulation. Therefore, targeting 14q32 cluster miRNAs may have therapeutic value in acquired BTK-resistant patients via regulation of the PTEN/AKT/mTOR signaling axis.Subject terms: Cancer, Chronic lymphocytic leukaemia  相似文献   

16.
The identification of proteins aberrantly expressed in malignant B-cells can potentially be used to develop new diagnostic, prognostic or therapeutic targets. Proteomic studies of B-cell malignancies have made significant progress, but further studies are needed to increase our coverage of the B-cell malignant proteome. To achieve this goal we stress the advantages of using sub-cellular fractionation, protein separation, quantitation and affinity purification techniques to identify hitherto unidentified signalling and regulatory proteins. For example, proteomic analysis of B-cell plasma membranes isolated from patients with mantle cell lymphoma (MCL) identified the voltage-gated proton channel (HVCN1,[1]). This protein has now been characterised as a key modulator of B-cell receptor (BCR) signalling and abrogation of HVCN1 function could have a role in the treatment of B-cell malignancies dependent on maintained BCR signalling [2]. Similarly, proteomic studies on cell lysates from prognostic subtypes of CLL, distinguished by the absence (UM-CLL) or presence (M-CLL) of somatic hypermutation of the immunoglobulin heavy chain locus identified nucleophosmin 1 (NMP1) as a potential prognostic marker [3,4]. Thus, targeted proteomic analysis on selected organelles or sub-cellular compartments can identify novel proteins with unexpected localisation or function in malignant B-cells that could be developed for clinical purposes.  相似文献   

17.
Bruton’s tyrosine kinase (BTK) has emerged as an attractive target related to B-lymphocytes dysfunctions, especially hematologic malignancies and autoimmune diseases. In our study, a series of diphenylaminopyrimidine derivatives bearing dithiocarbamate moieties were designed and synthesized as novel BTK inhibitors for treatment of B-cell lymphoma. Among all these compounds, 30ab (IC50 = 1.15 ± 0.19 nM) displays similar or more potent inhibitory activity against BTK than spebrutinib (IC50 = 2.12 ± 0.32 nM) and FDA approved drug ibrutinib (IC50 = 3.89 ± 0.57 nM), which is attributed to close binding of 30ab with BTK predicted by molecular docking. In particular, 30ab exhibits enhanced anti-proliferative activity against B-lymphoma cell lines at the IC50 concentration of 0.357 ± 0.02 μM (Ramos) and 0.706 ± 0.05 μM (Raji), respectively, almost 10-fold better than ibrutinib and spebrutinib. In addition, 30ab displays stronger selectivity on B-cell lymphoma over other cancer cell lines than spebrutinib. Furthermore, 30ab efficiently blocks BTK downstream pathways and results in apoptosis of cancer cells. In vivo xenograft model evaluation demonstrates the significant efficacy and broad safety margin of 30ab in treatment of B-cell lymphoma. We propose that compound 30ab is a candidate for further study and development based on our current findings.  相似文献   

18.
Bruton’s Tyrosine Kinase (BTK) and IL-2 Inducible T-cell Kinase (ITK) are enzymes responsible for the phosphorylation and activation of downstream effectors in the B-cell receptor (BCR) signaling and T cell receptor (TCR) signaling pathways, respectively. Ibrutinib is an FDA-approved potent inhibitor of both BTK and ITK that impairs B-cell and T-cell function. CD4 T cells and B cells are essential for the induction of chronic graft-versus-host disease (cGVHD). We evaluated these targets by testing the ability of Ibrutinib to prevent or ameliorate cGVHD, which is one of the major complications for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). We found that Ibrutinib significantly alleviated cGVHD across four different mouse models, accompanied by increased long-term survival and reduced clinical score. The clinical improvements in Ibrutinib-treated recipients were associated with decreased serum-autoantibodies, costimulatory molecule activation, B-cell proliferation, and glomerulonephritis compared to vehicle controls. Ibrutinib was also able to alleviate the clinical manifestations in acute GVHD (aGVHD), where the recipients were given grafts with or without B cells, suggesting that an inhibitory effect of Ibrutinib on T cells contributes to a reduction in both aGVHD and cGVHD pathogenesis. An effective prophylactic regimen is still lacking to both reduce the incidence and severity of human cGVHD following allo-HSCT. Our study shows that Ibrutinib is an effective prophylaxis against several mouse models of cGVHD with minimal toxicity and could be a promising strategy to combat human cGVHD clinically.  相似文献   

19.
CXCR5 [chemokine (C-X-C motif) receptor 5; also known as Burkitt lymphoma receptor 1 (BCR1)] is expressed on mature B-cells, subsets of CD4+ and CD8+ T-cells, and skin-derived migratory dendritic cells. Together with its ligand, CXCL13, CXCR5 is involved in guiding B-cells into the B-cell zones of secondary lymphoid organs as well as T-cell migration. This study evaluated the role of common germline genetic variation in CXCR5 in the risk and prognosis of non-Hodgkin lymphoma (NHL) using a clinic-based study of 1,521 controls and 2,694 NHL cases including 710 chronic lymphocytic leukemia/small lymphocytic lymphoma, 586 diffuse large B-cell lymphoma (DLBCL), 588 follicular lymphoma (FL), 137 mantle cell lymphoma (MCL), 230 marginal zone lymphoma (MZL), and 158 peripheral T-cell lymphoma (PTCL). Of the ten CXCR5 tag SNPs in our study, five were associated with risk of NHL, with rs1790192 having the strongest association (OR 1.19, 95 % CI 1.08–1.30; p = 0.0003). This SNP was most strongly associated with the risk of FL (OR 1.44, 95 % CI 1.25–1.66; p = 3.1 × 10?7), with a lower degree of association with DLBCL (OR 1.16, 95 % CI 1.01–1.33; p = 0.04) and PTCL (OR 1.29, 95 % CI 1.02–1.64; p = 0.04) but no association with the risk of MCL or MZL. For FL patients that were observed as initial disease management, the number of minor alleles of rs1790192 was associated with better event-free survival (HR 0.64; 95 % CI 0.47–0.87; p = 0.004). These results provide additional evidence for a role of host genetic variation in CXCR5 in lymphomagenesis, particularly for FL.  相似文献   

20.
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