B-cell Signaling Research Articles

Abstract 4568: Reversal of immune tolerance and increased anti tumoral immune response in a mouse model of CNS B cell lymphoma after combined XPO1 and BCR inhibition

Abstract Primary CNS lymphoma (PCNSL) is an aggressive non-Hodgkin lymphoma usually classified as ABC-DLBCL. Selinexor inactivates XPO-1 protein and induces anti-tumor effects mainly due to forced nuclear retention of tumor suppressors. It has shown excellent brain penetrance and promising results in glioblastoma and can inhibit both BCR and NF-kB signaling in malignant B-cells. Herein, we assessed the role of XPO-1 inhibition in intracerebral xenograft murine models. Proliferation and survival analysis in DLBCL cell lines showed that cell of origin does not influence sensitivity to selinexor and a strong synergy with ibrutinib mainly in ABC-DLBCL. We then established an orthotopic xenograft model of PCNSL by stereotactic injection of OCI-Ly10 cells transfected with luciferase into the brain parenchyma of nude athymic mice. Mice were treated with 5mg/kg oral selinexor or vehicle three times a week and bioluminiscence was assessed twice a week. Selinexor significantly blocked tumoral growth with differences starting as soon as 12 days after treatment. Also, selinexor improved mice survival (median: 48 days vs. 34 days). Next, we evaluated the potential synergy between ibrutinib and selinexor in vivo. For that mice were assigned into one of the following groups: selinexor (5mg/kg twice a week), ibrutinib (25mg/kg daily), combination or vehicle. The combination of both drugs significantly increased survival compared to both treatments as single agent, whereas there was no significant difference between ibrutinib and selinexor alone. Tumoral growth was equally blocked by all treatments. We next evaluated the innate immune response to the lymphoma by flow cytometry. Remarkably, CNS lymphomas were infiltrated by PD-1 positive M2 macrophages, as well as NK cells. Treatment with selinexor was able to decrease the proportion of M2-protumoral macrophages by half whereas the combination induced a 3-fold reduction (V: 22.72%+/-1.6, S: 12.29%+/-1.38, C: 7.1%+/-1.19). As a consequence, the proportion of M1 macrophages increased (V: 70.22%+/-1.8, S: 82.4%+/-1.7, C: 86.59%+/-1.5). In turn, ibrutinib was the main responsible for downregulation of PD-L1 in malignant cells (V: 3.7%+/-0.68, I: 0.6%+/-0.19, C: 0.19%+/- 0.08). Only when both treatments were combined there was significant downregulation of PD1 expression in M2 (V: 37.6%+/-5.5, C: 24.61%+/- 2.2) and a downregulation in PD1/SIRPα double positive M2 (V: 14.4%+-/1.44, C: 9.16%+/- 0.78). Selinexor and combination were able to significantly increase the proportion of NK cells (V:4.8%+/-0.21, S:9.7%+/-1.16, C:11.54%+/-2.02) and downregulate their PD1 expression (V:8.2%+/-2.7, S:3.7%+/-0.9, C: 2.8%+/-1.06). These results provide pre-clinical evidence for the development of selinexor and ibrutinib combination as new therapeutic option for PCNSL or DLBCL with CNS involvement. Citation Format: Marta Crespo, Isabel Jiménez, Júlia Carabia, Sabela Bobillo, Pau Abrisqueta, Carles Palacio, Juan Camilo Nieto, Joan Boix, Cecilia del Carmen Carpio, Josep Castellví, Joan Seoane, Francesc Bosch. Reversal of immune tolerance and increased anti tumoral immune response in a mouse model of CNS B cell lymphoma after combined XPO1 and BCR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4568.

Abstract 2144: Enhanced expression of β-catenin and Axl receptor tyrosine kinase in chronic lymphocytic leukemia (CLL) B-cells with co-culture on marrow stromal cells: Implications for drug resistance

Abstract Our study in CLL delineated a unique mechanism of how bone marrow stromal cells (BMSCs), may influence CLL B-cell signaling and drug resistance via modifying β-catenin and Axl status in leukemic cells. Purified primary CLL B-cells (n= 31) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=23) or CLL patients (n=12) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and real-time PCRs. We observed significant increases in expression of Axl at both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in co-cultured CLL B-cells. But, we did not see any significant change in β-catenin or Axl expression in BMSCs co-cultured with CLL B-cells. The CLL B-cells from co-culture had an upregulation in downstream P-ERK-1/2 but no change in P-AKT(Ser473). High nuclear active-β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β(Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β(Ser9) suggests inactivation of GSK-3β and β-catenin accumulation in co-cultured CLL B-cells. Additionally, we determined the phosphorylation status of Axl in CLL B-cells in co-culture compared with cultured alone. There was no change either at Y702 or total tyrosine phosphorylation in CLL B cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of Axl inhibitor TP-0903 on CLL B-cells in co-culture vs cultured alone. Interestingly, both Axl and β-catenin expression levels were further upregulated in CLL B-cells exposed to TP-0903 compared to CLL B-cells without TP-0903 in co-culture, indicating possible roles for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance. However, TP-0903 decreased P-Axl(Y702) as well as an Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells even in presence of BMSCs. This was associated with induction of cell death and overrode BMSC mediated protection. Treatments of fludarabine or chlorambucil also led to increase in expressions of both β-catenin and Axl in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. Here we show that stroma mediated increased expression in both β-catenin and Axl likely contribute to the survival and drug resistance of B-cells from patients with CLL. Understanding their role in drug metabolism is underway. Citation Format: Sutapa Sinha, Charla Secreto, Justin Boysen, Steven L. Warner, David J. Bearss, Asish Ghosh, Neil E. Kay. Enhanced expression of β-catenin and Axl receptor tyrosine kinase in chronic lymphocytic leukemia (CLL) B-cells with co-culture on marrow stromal cells: Implications for drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2144.

Subcellular topography modulates actin dynamics and signaling in B-cells.

B-cell signaling activation is most effectively triggered by the binding of B-cell receptors (BCRs) to membrane-bound antigens. In vivo, B-cells encounter antigen on antigen-presenting cells (APC), which possess complex surfaces with convoluted topographies, a fluid membrane and deformable cell bodies. However, whether and how the physical properties of antigen presentation affect B-cell activation is not well understood. Here we use nanotopographic surfaces that allow systematic variation of geometric parameters to show that surface features on a subcellular scale influence B-cell signaling and actin dynamics. Parallel nanoridges with spacings of 3 microns or greater induce actin intensity oscillations on the ventral cell surface. Nanotopography-induced actin dynamics requires BCR signaling, actin polymerization, and myosin contractility. The topography of the stimulatory surface also modulates the distribution of BCR clusters in activated B-cells. Finally, B-cells stimulated on nanopatterned surfaces exhibit intracellular calcium oscillations with frequencies that depend on topography. Our results point to the importance of physical aspects of ligand presentation, in particular, nanotopography for B-cell activation and antigen gathering.

Conjunctival transcriptome profiling of Solomon Islanders with active trachoma in the absence of Chlamydia trachomatis infection

BackgroundClinical signs of active (inflammatory) trachoma are found in many children in the Solomon Islands, but the majority of these individuals have no serological evidence of previous infection with Chlamydia trachomatis. In Temotu and Rennell and Bellona provinces, ocular infections with C. trachomatis were seldom detected among children with active trachoma; a similar lack of association was seen between active trachoma and other common bacterial and viral causes of follicular conjunctivitis. Here, we set out to characterise patterns of gene expression at the conjunctivae of children in these provinces with and without clinical signs of trachomatous inflammation-follicular (TF) and C. trachomatis infection.MethodsPurified RNA from children with and without active trachoma was run on Affymetrix GeneChip Human Transcriptome Array 2.0 microarrays. Profiles were compared between individuals with ocular C. trachomatis infection and TF (group DI; n = 6), individuals with TF but no C. trachomatis infection (group D; n = 7), and individuals without TF or C. trachomatis infection (group N; n = 7). Differential gene expression and gene set enrichment for pathway membership were assessed.ResultsConjunctival gene expression profiles were more similar within-group than between-group. Principal components analysis indicated that the first and second principal components combined explained almost 50% of the variance in the dataset. When comparing the DI group to the N group, genes involved in T-cell proliferation, B-cell signalling and CD8+ T cell signalling pathways were differentially regulated. When comparing the DI group to the D group, CD8+ T-cell regulation, interferon-gamma and IL17 production pathways were enriched. Genes involved in RNA transcription and translation pathways were upregulated when comparing the D group to the N group.ConclusionsGene expression profiles in children in the Solomon Islands indicate immune responses consistent with bacterial infection when TF and C. trachomatis infection are concurrent. The transcriptomes of children with TF but without identified infection were not consistent with allergic or viral conjunctivitis.

Mixed-species RNAseq analysis of human lymphoma cells adhering to mouse stromal cells identifies a core gene set that is also differentially expressed in the lymph node microenvironment of mantle cell lymphoma and chronic lymphocytic leukemia patients.

A subset of hematologic cancer patients is refractory to treatment or suffers relapse, due in part to minimal residual disease, whereby some cancer cells survive treatment. Cell-adhesion-mediated drug resistance is an important mechanism, whereby cancer cells receive survival signals via interaction with e.g. stromal cells. No genome-wide studies of in vitro systems have yet been performed to compare gene expression in different cell subsets within a co-culture and cells grown separately. Using RNA sequencing and species-specific read mapping, we compared transcript levels in human Jeko-1 mantle cell lymphoma cells stably adhered to mouse MS-5 stromal cells or in suspension within a co-culture or cultured separately as well as in stromal cells in co-culture or in separate culture. From 1050 differentially expressed transcripts in adherent mantle cell lymphoma cells, we identified 24 functional categories that together represent four main functional themes, anti-apoptosis, B-cell signaling, cell adhesion/migration and early mitosis. A comparison with previous mantle cell lymphoma and chronic lymphocytic leukemia studies, of gene expression differences between lymph node and blood, identified 116 genes that are differentially expressed in all three studies. From these genes, we suggest a core set of genes (CCL3, CCL4, DUSP4, ETV5, ICAM1, IL15RA, IL21R, IL4I1, MFSD2A, NFKB1, NFKBIE, SEMA7A, TMEM2) characteristic of cells undergoing cell-adhesion-mediated microenvironment signaling in mantle cell lymphoma/chronic lymphocytic leukemia. The model system developed and characterized here together with the core gene set will be useful for future studies of pathways that mediate increased cancer cell survival and drug resistance mechanisms.

Recent Research Advances on Signaling Pathway in B-cell Malignancy - Review

B-cell malignancy, a kind of malignant tumor of blood system, is characterized by heterogeneity and complexity of the pathogenesis. In this review, the recent advances of studies on B-cell malignancy and signaling pathways are briefly summarized. This review focuses on the role of signaling pathways, especially the NF-κ B, Wnt, PI3K/Akt, Notch and JAK/STAT signaling pathway in the occurrence and development of B-cell malignancy, and discussed the influence of signaling pathways on the invasiveness and drug resistance of B-cell malignancy, and the clinical application of signaling pathway inhibitors.

Single cell profiling of phospho-protein levels in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has a high incidence and a steeply growing prevalence in the Western world. The heterogeneity of the disease necessitates individual mapping of biology and predicted drug response in each patient as basis for administration of tailored treatments. Cell signaling aberrations may serve as biological indicators for suitable therapy. By applying phospho-specific flow cytometry, we mapped basal and induced phosphorylation levels of 20 phospho-epitopes on proteins relevant to B-cell signaling in B cells from 22 CLL patients and 25 normal controls. The signaling response of the cytostatic drugs fludarabine, doxorubicin and vincristine was also investigated. CLL cells exerted similar or lower basal phosphorylation levels compared to normal B cells, with the exception of STAT3 (pY705) which was increased. Interestingly, STAT3 inhibitors normalized the STAT3 (pY705) level and reduced cell viability. Vincristine treatment significantly modulated phosphorylation levels in CLL cells, while no effect was observed in controls or after fludarabine or doxorubicin treatment. After BCR stimulation, CLL cells showed a tendency towards impaired phosphorylation levels, significant for several of the analyzed proteins. However, the level of Akt (pS473) was more potently induced in IgHV unmutated CLL (UM-CLL) patient samples and was significantly higher than in M-CLL samples. Importantly, the PI3Kδ inhibitor idelalisib potently reversed the effect of anti-IgM on Akt (pS473). Thus, signaling aberrations could be identified by phosphoflow cytometry and aberrant signaling could be normalized by small molecule drugs. This approach can identify relevant drug targets as well as drug effects in the individual patient.

Introduction of pyrrolidineoxy or piperidineamino group at the 4-position of quinazoline leading to novel quinazoline-based phosphoinositide 3-kinase delta (PI3Kδ) inhibitors

Phosphoinositide 3-kinase Delta (PI3Kδ) plays a key role in B-cell signal transduction and inhibition of PI3Kδ was confirmed to have clinical benefit in certain types of activation of B-cell malignancies. Herein, we reported a novel series of 4-pyrrolidineoxy or 4-piperidineamino substituted quinazolines, showing potent PI3Kδ inhibitory activities. Among these compounds, 12d, 14b and 14c demonstrated higher potency against PI3Kδ with the half maximal inhibitory concentration (IC50) values of 4.5, 3.0, and 3.9 nM, respectively, which were comparable to idelalisib (IC50 = 2.7 nM). The further PI3K isoforms selectivity evaluation showed that compounds 12d, 14b and 14c have excellent PI3Kδ selectivity over PI3Kα, PI3Kβ, and PI3Kγ. Moreover, compounds 12d, 14b and 14c also displayed different anti-proliferative profiles against a panel of four human B cell lines including Ramos, Raji, RPMI-8226, and SU-DHL-6. The molecular docking simulation indicated several key hydrogen bonding interactions were formed. This study suggests the introduction of pyrrolidineoxy or piperidineamino groups into the 4-position of quinazoline leads to new potent and selective PI3Kδ inhibitors.

Improved biological insight and influence on management in indolent lymphoma. Talk 3: update on nodal and splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) and nodal marginal zone lymphoma (NMZL) are rare indolent chronic B-cell lymphomas. Prognosis is typically good with median survival around 10-15 years. Management is generally based on the presence of symptoms or high tumor burden. There are no standard treatments for these 2 entities, and therapeutic strategies are rapidly evolving. Clinical developments for these 2 entities are oriented by genomic studies, with largely overlapping mutational profiles involving the NOTCH, B-cell receptor (BcR) and nuclear factor κB (NF-κB) signaling, chromatin remodeling, and the cytoskeleton. Although new therapeutic options based on targeting signaling pathways and overcoming resistance are increasingly available, few specific prospective studies are performed for these rare subtypes, limiting the conclusions that can be drawn. Novel drugs targeting B-cell signaling have shown promise, including ibrutinib and copanlisib. The second-generation oral immunomodalator (IMiD) lenalidomide showed impressive results when combined with rituximab. Other potential solutions include targeting the NF-κB, JAK/STAT, BCL2, NOTCH, and Toll-like receptor signaling pathways; however, studies in these 2 MZL entities are yet to prove a definitive benefit. Molecular profiling is now a cornerstone of diagnostic, prognostic, and therapeutic strategies to offer patient- and disease-specific solutions. The development of a wider range of effective targeted therapies and prognostic biomarkers is keenly awaited and is expected to strongly affect the natural history of SMZL and NMZL.

Current and future therapies for SLE: obstacles and recommendations for the development of novel treatments.

SLE is a serious, debilitating autoimmune disease that affects various organs and body systems. Of all the heterogeneous autoimmune diseases, SLE is perhaps the most heterogeneous. Patients with SLE, who are primarily female, have diverse disease manifestations and severity. SLE is characterised by substantial concentrations of autoantibodies against nuclear antigens, which are thought to be caused by immune cell dysregulation. Until recently, several immunosuppressant agents were used to treat this disease. Efforts to develop drugs against targets potentially involved in disease mechanisms have resulted in the identification and use of BAFF (B-cell activating factor)/APRIL (a proliferation-inducing ligand) inhibitors to treat SLE. Drugs in late-stage development that focus on pathways that are dysregulated in SLE include those that target the interferon pathway, T-cell signalling and B-cell signalling. New therapeutic agents are still necessary because of the unmet medical needs associated with this disease, including insufficient disease control, poor health-related quality of life, comorbidities, toxicity of the majority of therapies and diminished survival. Despite the substantial long-term investment of research, clinical activity and resources for identifying new treatments for this disease, only one new therapy, the biological belimumab, has been approved in the past 50 years. Efforts to develop drugs to address these needs are challenged by problems associated with disease heterogeneity, variable disease mechanisms and trial design. This review provides an overview of current and future treatments, discusses challenges in the SLE drug development process and offers recommendations for overcoming these challenges.

Differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV).

Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae and is economically important to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius (BF), causing immunosuppression and morbidity in young chickens. In addition to strains that cause classical Gumboro disease, the so-called 'very virulent' (vv) strain, also in circulation, causes more severe disease and increased mortality. IBDV has traditionally been controlled through the use of live attenuated vaccines, with attenuation resulting from serial passage in non-lymphoid cells. However, the factors that contribute to the vv or attenuated phenotypes are poorly understood. In order to address this, we aimed to investigate host cell-IBDV interactions using a recently described chicken primary B-cell model, where chicken B cells are harvested from the BF and cultured ex vivo in the presence of chicken CD40L. We demonstrated that these cells could support the replication of IBDV when infected ex vivo in the laboratory. Furthermore, we evaluated the gene expression profiles of B cells infected with an attenuated strain (D78) and a very virulent strain (UK661) by microarray. We found that key genes involved in B-cell activation and signalling (TNFSF13B, CD72 and GRAP) were down-regulated following infection relative to mock, which we speculate could contribute to IBDV-mediated immunosuppression. Moreover, cells responded to infection by expressing antiviral type I IFNs and IFN-stimulated genes, but the induction was far less pronounced upon infection with UK661, which we speculate could contribute to its virulence.

Novel Mutations Involving NF-κB and B-Cell Signaling Pathways in Primary Cutaneous Large B-Cell Lymphoma, Leg-Type and Comparison with Sézary Syndrome

Multiple genomic mutations, especially those involving the NF-κB pathway, have been characterized in primary cutaneous large B-cell lymphoma, leg type. However, its genomic profiling remains limited given its rarity. In a recent study, Mareschal etal. performed next-generation sequencing and whole-exome sequencing, identifying new driver genes while also confirming the role of myeloid differentiation primary response gene 88 in the molecular pathogenesis of the disease.

A Small Molecule Inhibitor of Bruton's Tyrosine Kinase Involved in B-Cell Signaling.

Protein kinases are fundamental within almost all cellular signal transduction networks. Among these, Bruton’s tyrosine kinase (Btk), which belongs to the Tec family of proteins, plays an imperative part in B-cell signaling. Owing to its role, Btk has been established as an important therapeutic target for a vast range of disorders related to B-cell development and function, such as the X-linked agammaglobulinemia, various B-cell malignancies, inflammation, and autoimmune diseases. Herein, using computer-based screening of a library of 20 million small molecules, we identified a small molecule capable of directly binding the Btk kinase domain. On the basis of this hit compound, we conducted a focused structure-similarity search to explore the effect of different chemical modifications on binding toward Btk. This search identified the molecule N2,N6-bis(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-9H-purine-2,6-diamine as a potent inhibitor of Btk. The latter small molecule binds Btk with a dissociation constant of 250 nM and inhibits Btk activity both in vitro and in-cell.

TLR7/TLR9- and B Cell Receptor-Signaling Crosstalk: Promotion of Potentially Dangerous B Cells.

B cells are capable of receptor-mediated responses to foreign antigens. Recognition of microbial-derived nucleic acid (NA) by toll-like receptors (TLRs) 7 and 9 in B cells has been substantiated. Endogenous NA released from damaged or dying cells can also be immunogenic in certain contexts and can incite aberrant activation of B cells. When TLR-driven B cell receptor (BCR)-activated B cells are not properly constrained, pathologic autoantibodies are produced. It is also clear that endosomal TLR7/TLR9 can operate in conjunction with BCR. In addition to BCR signaling, a balance between TLR7 and TLR9 is pivotal in the development of B cell autoreactivity. While TLR9 is important in normal memory B cell responses through BCR, TLR9 activation has been implicated in autoantibody production. Paradoxically, TLR9 also plays known protective roles against autoimmunity by directly and indirectly inhibiting TLR7-mediated autoantibody production. Herein, we summarize literature supporting mechanisms underpinning the promotion of pathological BCR-activated B cells by TLR7 and TLR9. We focus on the literature regarding known points of TLR7/TLR9 and BCR crosstalk. Data also suggest that the degree of TLR responsiveness relies on alterations of certain intrinsic B-cell signaling molecules and is also context specific. Because allogeneic hematopoietic stem cell transplantation is a high NA and B cell-activating factor environment, we conclude that B cell studies of synergistic TLR–BCR signaling in human diseases like chronic graft-versus-host disease are warranted. Further understanding of the distinct molecular pathways mediating TLR–BCR synergy will lead to the development of therapeutic strategies in autoimmune disease states.

Abstract 4082: Time- and exposure-dependent pharmacodynamic changes induced by the BTK inhibitor GS-4059 in healthy subjects

Abstract Bruton’s Tyrosine Kinase (BTK) plays an important role in B-cell signaling, cell proliferation and survival and is an established drug target in B-cell malignancies. GS-4059 (ONO-4059) is an irreversible, small molecule inhibitor of BTK, which is in development for hematological malignancies and rheumatoid arthritis. A Phase 1 study to evaluate the effect of organic anion transporting polypeptide (OATP) 1B1 and 1B3 inhibitors and inducers of cytochrome P450 3A (CYP3A) on GS-4059 Pharmacokinetics (PK) was performed in healthy subjects (N=15). Subjects were administered a single dose of 100 mg GS-4059 alone or in combination with a single or multiple doses of rifampin (600mg) and blood samples were collected up to 48 hours post dose to evaluate PK and Pharmacodynamic (PD) markers in peripheral blood mononuclear cells (PBMCs). PD biomarkers were exploratory endpoints and are reported here. PD was evaluated using the BTK occupancy assay and the Basophil Activation Test (BAT). A novel duplex homogeneous BTK occupancy assay was used to quantitatively measure GS-4059 binding to human BTK to assess target coverage. The assay is based on Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) and simultaneously measures the level of free BTK as well as total BTK protein in PBMCs collected from subjects in the study. Free BTK levels in PBMCs decreased rapidly on treatment with GS-4059 with no detectable free BTK 3-24 hours post dose and no notable changes in total BTK. All subjects had free BTK levels below the limit of detection (LOD 6.8 ng/mL) 3-24 hours after dosing. Free BTK levels started to increase after 48 hours. Free BTK levels on treatment with multiple doses of rifampin plus GS-4059 followed a similar trend with no free BTK detectable 3-6 hours post dose but resulted in shorter duration of inhibition and more rapid recovery of free BTK levels. GS-4059 plasma exposure (Cmax and AUC) was ~70% lower after multiple doses of rifampin, due to a drug-drug interaction. As a second PD marker, inhibition of CD63+ basophil activation in the BAT was measured. In this functional assay the median inhibition of CD63+ basophil activation was between 90% and 100% at the time of maximum inhibition (3 hours post dose). At the peak of inhibition 13 of the 14 subjects had inhibition of basophil activation higher than 97%. In line with the BTK occupancy data, inhibition of CD63+ basophil activation was significantly lower with reduced GS-4059 plasma exposure. BTK occupancy as a direct measure of target binding and the functional assay measuring the inhibition of basophil activation showed a similar time course with maximum effect in both assays achieved at 3-6 hours post dose. These data demonstrate that a single 100 mg dose of GS-4059 results in full BTK occupancy in healthy subject PBMCs as well as functional changes measured in basophil activation as a surrogate for BTK signaling pathway inhibition. Citation Format: Juliane M. Jürgensmeier, Hoa Truong, Lianqing Zheng, Yuanyuan Xiao, Cara Nelson, Siddhartha Mitra, Andrew N. Billin, Helen Yu. Time- and exposure-dependent pharmacodynamic changes induced by the BTK inhibitor GS-4059 in healthy subjects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4082. doi:10.1158/1538-7445.AM2017-4082

Identification of Somatic Mutations in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type by Massive Parallel Sequencing

To determine whether the mutational profile of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) is unique by comparison with other diffuse large B-cell lymphoma subtypes, we analyzed a total cohort of 20 PCLBCL-LT patients by using next-generation sequencing with a lymphoma panel designed for diffuse large B-cell lymphoma. We also analyzed 12 pairs of tumor and control DNA samples by whole-exome sequencing, which led us to perform resequencing of three selected genes not included in the lymphoma panel: TBL1XR1, KLHL6, and IKZF3. Our study clearly identifies an original mutational landscape of PCLBCL-LT with a very restricted set of highly recurrent mutations (>40%) involving MYD88 (p.L265P variant), PIM1, and CD79B. Other genes involved in B-cell signaling, NF-κB activation, or DNA modeling were found altered, notably TBL1XR1 (33%), MYC (26%) CREBBP (26%), and IRF4 (21%) or HIST1H1E (41%). MYD88L265P variant was associated with copy number variations or copy neutral loss of heterozygosity in 60% of patients. The most frequent genetic losses involved CDKN2A/2B, TNFAIP3/A20, PRDM1, TCF3, and CIITA. Together, these results show that PCLBCL-LT exhibits a unique mutational landscape, combining highly recurrent hotspot mutations in genes involved in NF-kB and B-cell signaling pathways, which provides a rationale for using selective inhibitors of the B-cell receptor.

Molekularna patogeneza przewlekłej białaczki limfocytowej

Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia, characterized by accumulation of mature but functionally incompetent clonal B lymphocytes in peripheral blood, bone marrow and lymphoid tissues. The clinical course of CLL varies from patients with indolent, stable disease to those with aggressive leukemia who succumb to their disease in a short time. The use of novel molecular biology techniques revealed genetic and epigenetic heterogeneity among CLL patients and allowed to define novel somatic mutations of prognostic value. The CLL genome and epigenome undergo dynamic changes during disease course due to clonal evolution, which leads to selection and expansion of leukemic clones with the highest survival potential. This review focuses on the key aspects of CLL molecular pathogenesis including genetic and epigenetic alterations, B-cell signaling and the role of tumor microenvironment. Progress in the understanding of CLL biology will help to develop more accurate prognostication models and enable more personalized patient treatment in the future.

B-cell tolerance and autoimmunity.

Self-reactive B cells are tolerized at various stages of B-cell development and differentiation, including the immature B-cell stage (central tolerance) and the germinal center (GC) B-cell stage, and B-cell tolerance involves various mechanisms such as deletion, anergy, and receptor editing. Self-reactive B cells generated by random immunoglobulin variable gene rearrangements are tolerized by central tolerance and anergy in the periphery, and these processes involve apoptosis regulated by Bim, a pro-apoptotic member of the Bcl-2 family, and regulation of B-cell signaling by various phosphatases, including SHIP-1 and SHP-1. Self-reactive B cells generated by somatic mutations during GC reaction are also eliminated. Fas is not directly involved in this process but prevents persistence of GC reaction that allows generation of less stringently regulated B cells, including self-reactive B cells. Defects in self-tolerance preferentially cause lupus-like disease with production of anti-nuclear antibodies, probably due to the presence of a large potential B-cell repertoire reactive to nucleic acids and the presence of nucleic acid-induced activation mechanisms in various immune cells, including B cells and dendritic cells. A feed-forward loop composed of anti-nuclear antibodies produced by B cells and type 1 interferons secreted from nucleic acid-activated dendritic cells plays a crucial role in the development of systemic lupus erythematosus.

Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

Activating mutations of NOTCH1 (a well-known oncogene in T-cell acute lymphoblastic leukemia) are present in ∼4-13% of chronic lymphocytic leukemia (CLL) cases, where they are associated with disease progression and chemorefractoriness. However, the specific role of NOTCH1 in leukemogenesis remains to be established. Here, we report that the active intracellular portion of NOTCH1 (ICN1) is detectable in ∼50% of peripheral blood CLL cases lacking gene mutations. We identify a "NOTCH1 gene-expression signature" in CLL cells, and show that this signature is significantly enriched in primary CLL cases expressing ICN1, independent of NOTCH1 mutation. NOTCH1 target genes include key regulators of B-cell proliferation, survival, and signal transduction. In particular, we show that NOTCH1 transactivates MYC via binding to B-cell-specific regulatory elements, thus implicating this oncogene in CLL development. These results significantly extend the role of NOTCH1 in CLL pathogenesis, and have direct implications for specific therapeutic targeting.

Receptor-guided 3D-QSAR studies, molecular dynamics simulation and free energy calculations of Btk kinase inhibitors

BackgroundBruton tyrosine kinase (Btk) plays an important role in B-cell development, differentiation, and signaling. It is also found be in involved in male immunodeficiency disease such as X-linked agammaglobulinemia (XLA). Btk is considered as a potential therapeutic target for treating autoimmune diseases and hematological malignancies.ResultsIn this work, a combined molecular modeling study was performed on a series of thieno [3,2-c] pyridine-4-amine derivatives as Btk inhibitors. Receptor-guided COMFA (q2 = 0.574, NOC = 3, r2 = 0.924) and COMSIA (q2 = 0.646, NOC = 6, r2 = 0.971) models were generated based on the docked conformation of the most active compound 26. All the developed models were tested for robustness using various validation techniques. Furthermore, a 5-ns molecular dynamics (MD) simulation and binding free energy calculations were carried out to determine the binding modes of the inhibitors and to identify crucial interacting residues. The rationality and stability of molecular docking and 3D-QSAR results were validated by MD simulation. The binding free energies calculated by the MM/PBSA method showed the importance of the van der Waals interaction.ConclusionsA good correlation between the MD results, docking studies, and the contour map analysis were observed. The study has identified the key amino acid residues in Btk binding pocket. The results from this study can provide some insights into the development of potent, novel Btk inhibitors.