Francisella tularensis is a facultative intracellular, gram-negative bacterium that induces apoptosis in macrophages and B cells. Here we show apoptotic pathways that are activated in the Ramos human B cell line in the course of F. tularensis infection. Live bacteria F. tularensis FSC200 activate caspases 8, 9 and 3, as well as Bid; release cytochrome c and apoptosis-inducing factor from mitochondria; and induce depolarization of mitochondrial membrane potential in the Ramos cell line, thus leading these cells to apoptosis. Unlike live bacteria, killed F. tularensis FSC200 bacteria activated only caspase 3, and did not cause apoptosis of Ramos cells as measured by annexin V. Killed bacteria also caused accumulation of anti-apoptotic protein Bclx(L) in mitochondrial membranes. Thus, live F. tularensis activates both caspase pathways (receptor-mediated and intrinsic) as well as caspase-independent mitochondrial death.

• MeSH
• apoptóza * MeSH
• B-lymfocyty mikrobiologie   MeSH
• buněčné linie MeSH
• cytochromy c metabolismus   MeSH
• Francisella tularensis patogenita   MeSH
• kaspasa 3 biosyntéza   MeSH
• kaspasa 8 biosyntéza   MeSH
• kaspasa 9 biosyntéza   MeSH
• lidé MeSH
• membránový potenciál mitochondrií MeSH
• mitochondrie enzymologie   fyziologie   MeSH
• protein Bid biosyntéza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BID protein, human MeSH Prohlížeč
• CASP8 protein, human MeSH Prohlížeč
• cytochromy c MeSH
• kaspasa 3 MeSH
• kaspasa 8 MeSH
• kaspasa 9 MeSH
• protein Bid MeSH

Immunity to Francisella tularensis is largely mediated by T lymphocytes but an important role of B lymphocytes in early stage of infection was previously uncovered. We wanted to find out if F. tularensis is able to infect B cells and/or influence them by direct contact. To investigate this possibility we infected B cell lines from mouse (A20) or humans (Ramos RA-1), or primary mouse spleen cells, with F. tularensis LVS and F. tularensis FSC200 in vitro. In all cases, we detected bacteria on the cell surface and inside the B cells using transmission electron microscopy. More than 20% cells were infected by microbes after 24h. The number of bacteria, determined by CFU, increased about 1 log during 24h. Infection with live bacteria led to apoptosis of Ramos cells and mouse CD19(+) spleen cells. Approximately 30% of cells were apoptotic after 24h and 70% after 48 h, independently of the F. tularensis strain, while only 10% of non-infected cell were apoptotic at either time point. Apoptosis was confirmed by Western blot using anti-PARP antibodies. Thus, this study demonstrates unique phenomenon - namely, the ability of the intracellular pathogen F. tularensis to invade and induce apoptosis in B cells.

• MeSH
• apoptóza * MeSH
• B-lymfocyty imunologie   mikrobiologie   fyziologie   MeSH
• buněčné linie MeSH
• Francisella tularensis imunologie   fyziologie   MeSH
• lidé MeSH
• myši MeSH
• transmisní elektronová mikroskopie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Secondary diversification of the Ig repertoire occurs through somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR)-three processes that are initiated by activation-induced cytidine deaminase (AID). AID targets Ig genes at orders of magnitude higher than the rest of the genome, but the basis for this specificity is poorly understood. We have previously demonstrated that enhancers and enhancer-like sequences from Ig genes are capable of stimulating SHM of neighboring genes in a capacity distinct from their roles in increasing transcription. Here, we use an in vitro proteomics approach to identify E-box, MEF2, Ets, and Ikaros transcription factor family members as potential binders of these enhancers. ChIP assays in the hypermutating Ramos B cell line confirmed that many of these factors bound the endogenous Igλ enhancer and/or the IgH intronic enhancer (Eμ) in vivo. Further investigation using SHM reporter assays identified binding sites for E2A and MEF2B in Eμ and demonstrated an association between loss of factor binding and decreases in the SHM stimulating activity of Eμ mutants. Our results provide novel insights into trans-acting factors that dictate SHM targeting and link their activity to specific DNA binding sites within Ig enhancers.

• Klíčová slova
• AID, E2A, MEF2B, Ramos B cell line, Somatic hypermutation,
• MeSH
• geny pro imunoglobuliny MeSH
• kur domácí MeSH
• lidé MeSH
• somatická hypermutace imunoglobulinových genů fyziologie   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• transkripční faktory MeSH

Bruton's Tyrosine Kinase (BTK) is a key driver of hematological malignancies, autoimmune disorders, and neuroinflammation, making it an attractive therapeutic target. Proteolysis targeting chimeras (PROTACs) offer a novel strategy for BTK degradation via the E3 ubiquitin ligase pathway. Here, we evaluated nine azaspirooxindolinone-based PROTAC derivatives for their cytotoxicity and BTK-targeting activity. Several compounds exhibited potent cytotoxicity against BTK-high RAMOS lymphoma cells without affecting non-cancer fibroblasts or normal T/B-cell lymphocytes. Among them, PROTAC 25 emerged as the most effective degraded, achieving a Dmax of 72.84 % and DC50 of 0.27 μM in a proteasome-dependent manner. Although PROTAC 25 was cytotoxic to IL-2-inducible T cell Kinase (ITK)-positive cells, ITK protein levels remained unaffected. Furthermore, kinase assays revealed that PROTAC 25 inhibited BTK kinase activity (IC₅₀ = 0.44 μM) with moderate selectivity over ITK (IC₅₀ = 2.16 μM). Notably, PROTAC 25 suppressed BTK-mediated downstream signaling in RAMOS cells, as evidenced by reduced phosphorylation of BTK and its downstream effector, p38 MAPK. These findings highlight PROTAC 25 as a promising BTK degrader with therapeutic potential and underscore the value of azaspirooxindolinone-based PROTACs in targeting BTK-driven diseases.

• Klíčová slova
• Antiproliferative activity, Azaspirooxindolinone, Bruton's tyrosine kinase, IL-2-inducible T-cell kinase, Jurkat, PROTAC, Ramos,
• MeSH
• inhibitory proteinkinas farmakologie   chemie   chemická syntéza   MeSH
• lidé MeSH
• molekulární struktura MeSH
• nádorové buněčné linie MeSH
• oxindoly farmakologie   chemie   chemická syntéza   MeSH
• proliferace buněk účinky léků   MeSH
• proteinkinasa BTK * antagonisté a inhibitory   metabolismus   MeSH
• proteolýza * účinky léků   MeSH
• protinádorové látky * farmakologie   chemie   chemická syntéza   MeSH
• screeningové testy protinádorových léčiv MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• BTK protein, human MeSH Prohlížeč
• inhibitory proteinkinas MeSH
• oxindoly MeSH
• proteinkinasa BTK * MeSH
• protinádorové látky * MeSH

BACKGROUND: Despite recent advances in the treatment of aggressive lymphomas, a significant fraction of patients still succumbs to their disease. Thus, novel therapies are urgently needed. As the anti-CD20 antibody rituximab and the CD19-targeting antibody tafasitamab share distinct modes of actions, we investigated if dual-targeting of aggressive lymphoma B-cells by combining rituximab and tafasitamab might increase cytotoxic effects. METHODS: Antibody single and combination efficacy was determined investigating different modes of action including direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in in vitro and in vivo models of aggressive B-cell lymphoma comprising diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). RESULTS: Three different sensitivity profiles to antibody monotherapy or combination treatment were observed in in vitro models: while 1/11 cell lines was primarily sensitive to tafasitamab and 2/11 to rituximab, the combination resulted in enhanced cell death in 8/11 cell lines in at least one mode of action. Treatment with either antibody or the combination resulted in decreased expression of the oncogenic transcription factor MYC and inhibition of AKT signaling, which mirrored the cell line-specific sensitivities to direct cytotoxicity. At last, the combination resulted in a synergistic survival benefit in a PBMC-humanized Ramos NOD/SCID mouse model. CONCLUSION: This study demonstrates that the combination of tafasitamab and rituximab improves efficacy compared to single-agent treatments in models of aggressive B-cell lymphoma in vitro and in vivo.

• Klíčová slova
• CD19, CD20, antibody therapy, lymphoma, rituximab, tafasitamab, tumor immunology,
• MeSH
• Burkittův lymfom * farmakoterapie   MeSH
• difúzní velkobuněčný B-lymfom * farmakoterapie   MeSH
• humanizované monoklonální protilátky MeSH
• leukocyty mononukleární MeSH
• myši inbrední NOD MeSH
• myši SCID MeSH
• myši MeSH
• rituximab farmakologie   terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• humanizované monoklonální protilátky MeSH
• rituximab MeSH
• tafasitamab MeSH Prohlížeč

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

• MeSH
• AICDA (aktivací indukovaná cytidindeamináza) MeSH
• aktivace lymfocytů MeSH
• Burkittův lymfom genetika   imunologie   MeSH
• cytidindeaminasa genetika   MeSH
• genetická transkripce MeSH
• imunoglobuliny genetika   metabolismus   MeSH
• kur domácí MeSH
• lidé MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• podskupiny B-lymfocytů imunologie   MeSH
• ptačí proteiny genetika   metabolismus   MeSH
• RNA-polymerasa II genetika   metabolismus   MeSH
• rozmanitost protilátek MeSH
• somatická hypermutace imunoglobulinových genů MeSH
• zesilovače transkripce genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• AICDA (aktivací indukovaná cytidindeamináza) MeSH
• cytidindeaminasa MeSH
• imunoglobuliny MeSH
• ptačí proteiny MeSH
• RNA-polymerasa II MeSH

A large body of evidence suggests that hypoxia drives aggressive molecular features of malignant cells irrespective of cancer type. Non-Hodgkin lymphomas (NHL) are the most common hematologic malignancies characterized by frequent involvement of diverse hypoxic microenvironments. We studied the impact of long-term deep hypoxia (1% O2) on the biology of lymphoma cells. Only 2 out of 6 tested cell lines (Ramos, and HBL2) survived ≥ 4 weeks under hypoxia. The hypoxia-adapted (HA)b Ramos and HBL2 cells had a decreased proliferation rate accompanied by significant suppression of both oxidative phosphorylation and glycolytic pathways. Transcriptome and proteome analyses revealed marked downregulation of genes and proteins of the mitochondrial respiration complexes I and IV, and mitochondrial ribosomal proteins. Despite the observed suppression of glycolysis, the proteome analysis of both HA cell lines showed upregulation of several proteins involved in the regulation of glucose utilization including the active catalytic component of prolyl-4-hydroxylase P4HA1, an important druggable oncogene. HA cell lines demonstrated increased transcription of key regulators of auto-/mitophagy, e.g., neuritin, BCL2 interacting protein 3 (BNIP3), BNIP3-like protein, and BNIP3 pseudogene. Adaptation to hypoxia was further associated with deregulation of apoptosis, namely upregulation of BCL2L1/BCL-XL, overexpression of BCL2L11/BIM, increased binding of BIM to BCL-XL, and significantly increased sensitivity of both HA cell lines to A1155463, a BCL-XL inhibitor. Finally, in both HA cell lines AKT kinase was hyperphosphorylated and the cells showed increased sensitivity to copanlisib, a pan-PI3K inhibitor. In conclusion, our data report on several shared mechanisms of lymphoma cell adaptation to long-term hypoxia including: 1. Upregulation of proteins responsible for glucose utilization, 2. Degradation of mitochondrial proteins for potential mitochondrial recycling (by mitophagy), and 3. Increased dependence on BCL-XL and PI3K-AKT signaling for survival. In translation, inhibition of glycolysis, BCL-XL, or PI3K-AKT cascade may result in targeted elimination of HA lymphoma cells.

• Publikační typ
• časopisecké články MeSH

Interleukin-2-inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK) are two important members of the Tec family with crucial roles in immune system function. Deregulation in ITK and BTK activity is linked to several hematological malignancies, making them key targets for cancer immunotherapy. In this study, we synthesized a series of azaspirooxindolinone derivatives and evaluated their cytotoxic activity against ITK/BTK-negative and positive cancer cell lines, followed by enzymatic inhibition studies to assess the ITK/BTK kinase selectivity of two hit compounds. Several compounds demonstrated selective cytotoxicity against ITK- or BTK-expressing cells. Compound 3d exhibited high cytotoxicity in ITK-positive Jurkat (IC50 = 3.58 µM) and BTK-positive Ramos (IC50 = 3.06 µM) cells, while compound 3j showed strong cytotoxicity in Ramos (IC50 = 1.38 µM) and Jurkat (IC50 = 4.16 µM) cells. Compounds 3a and 3e were selectively cytotoxic in Jurkat cells (IC50 = 9.36 µM and 10.85 µM, respectively), while compounds 3f and 3g were highly cytotoxic in Ramos cells (IC50 = 1.82 µM and 1.42 µM, respectively). None of the active compounds exhibited cytotoxicity in non-cancer cell lines (IC50 > 50 µM), demonstrating their selectivity for malignant cells. Enzyme inhibition assay showed that 3d is a selective ITK inhibitor (IC50 = 0.91 µM) with no detectable BTK inhibition, aligning with its strong activity in ITK-positive cells. In contrast, compound 3j did not inhibit ITK or BTK enzymatically, suggesting an alternative mechanism of action. These findings highlight 3d as a promising ITK inhibitor and warrant further investigation to elucidate its mechanism of action.

• Klíčová slova
• Anti-cancer derivatives, Azaspirooxindolinones, Bruton’s tyrosine kinase, Interleukin-2-inducible T-cell kinase, Molecular docking,
• MeSH
• inhibitory proteinkinas * farmakologie   chemická syntéza   chemie   MeSH
• lidé MeSH
• molekulární struktura MeSH
• nádorové buněčné linie MeSH
• oxindoly farmakologie   chemie   chemická syntéza   MeSH
• proliferace buněk účinky léků   MeSH
• proteinkinasa BTK * antagonisté a inhibitory   metabolismus   MeSH
• protinádorové látky * farmakologie   chemická syntéza   chemie   MeSH
• racionální návrh léčiv * MeSH
• screeningové testy protinádorových léčiv * MeSH
• simulace molekulového dockingu MeSH
• spirosloučeniny chemie   farmakologie   chemická syntéza   MeSH
• tyrosinkinasy * antagonisté a inhibitory   metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• BTK protein, human MeSH Prohlížeč
• emt protein-tyrosine kinase MeSH Prohlížeč
• inhibitory proteinkinas * MeSH
• oxindoly MeSH
• proteinkinasa BTK * MeSH
• protinádorové látky * MeSH
• spirosloučeniny MeSH
• tyrosinkinasy * MeSH

Bruton's tyrosine kinase (BTK) is a promising molecular target for several human B-cell-related autoimmune disorders, inflammation, and haematological malignancies. The pathogenic alterations in various cancer tissues depend on mutant BTK for cell proliferation and survival, and BTK is also overexpressed in a range of hematopoietic cells. Due to this, BTK is emerging as a potential drug target to treat various human diseases, and several reversible and irreversible inhibitors have been developed and are being developed. As a result, BTK inhibition, clinically validated as an anticancer treatment, is finding great interest in B-cell malignancies and solid tumours. This study focuses on the design and synthesis of new oxindole sulfonamide derivatives as promising inhibitors of BTK with negligible off-target effects. The most cytotoxic compounds with greater basicity were PID-4 (2.29±0.52 μM), PID-6 (9.37±2.47 μM), and PID-19 (2.64±0.88 μM). These compounds caused a selective inhibition of Burkitt's lymphoma RAMOS cells without significant cytotoxicity in non-BTK cancerous and non-cancerous cell lines. Further, PID-4 showed promising activity in inhibiting BTK and downstream signalling cascades. As a potent inhibitor of Burkitt's lymphoma cells, PID-4 is a promising lead for developing novel chemotherapeutics.

• Klíčová slova
• Antiproliferation, B-cell malignancies, Bruton's tyrosine kinase, Cytotoxicity, Knoevenagel condensation, Oxindole sulfonamide,
• MeSH
• Burkittův lymfom * farmakoterapie   MeSH
• inhibitory proteinkinas MeSH
• inhibitory tyrosinkinasy * MeSH
• lidé MeSH
• proteinkinasa BTK MeSH
• sulfonamidy farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory proteinkinas MeSH
• inhibitory tyrosinkinasy * MeSH
• proteinkinasa BTK MeSH
• sulfonamidy MeSH

Bruton's tyrosine kinase (BTK) is a non-RTK cytoplasmic kinase predominantly expressed by hemopoietic lineages, particularly B-cells. A new oxindole-based focused library was designed to identify potent compounds targeting the BTK protein as anticancer agents. This study used rational approaches like structure-based pharmacophore modeling, docking, and ADME properties to select compounds. Molecular dynamics simulations carried out at 20 ns supported the stability of compound 9g within the binding pocket. All the compounds were synthesized and subjected to biological screening on two BTK-expressing cancer cell lines, RAMOS and K562; six non-BTK cancer cell lines, A549, HCT116 (parental and p53-/-), U2OS, JURKAT, and CCRF-CEM; and two non-malignant fibroblast lines, BJ and MRC-5. This study resulted in the identification of four new compounds, 9b, 9f, 9g, and 9h, possessing free binding energies of -10.8, -11.1, -11.3, and -10.8 kcal/mol, respectively, and displaying selective cytotoxicity against BTK-high RAMOS cells. Further analysis demonstrated the antiproliferative activity of 9h in RAMOS cells through selective inhibition of pBTK (Tyr223) without affecting Lyn and Syk, upstream proteins in the BCR signaling pathway. In conclusion, we identified a promising oxindole derivative (9h) that shows specificity in modulating BTK signaling pathways.

• Publikační typ
• časopisecké články MeSH