Toll-like receptors (TLRs) recognize molecular patterns preferentially expressed by pathogens. In endosomes, TLR9 is activated by unmethylated bacterial DNA, resulting in proinflammatory cytokine secretion via the adaptor protein MyD88. We demonstrate that CpG oligonucleotides activate a TLR9-independent pathway initiated by two Src family kinases, Hck and Lyn, which trigger a tyrosine phosphorylation–mediated signaling cascade. This cascade induces actin cytoskeleton reorganization, resulting in cell spreading, adhesion, and motility. CpG-induced actin polymerization originates at the plasma membrane, rather than in endosomes. Chloroquine, an inhibitor of CpG-triggered cytokine secretion, blocked TLR9/MyD88-dependent cytokine secretion as expected but failed to inhibit CpG-induced Src family kinase activation and its dependent cellular responses. Knock down of Src family kinase expression or the use of specific kinase inhibitors blocked MyD88-dependent signaling and cytokine secretion, providing evidence that tyrosine phosphorylation is both CpG induced and an upstream requirement for the engagement of TLR9. The Src family pathway intersects the TLR9–MyD88 pathway by promoting the tyrosine phosphorylation of TLR9 and the recruitment of Syk to this receptor.

BackgroundThe CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL.MethodsPatient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model.ResultsPF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine.ConclusionsWe show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-017-0435-x) contains supplementary material, which is available to authorized users.

Cathepsin S is considered crucial for normal presentation of major histocompatibility complex (MHC) class II-restricted antigens by antigen presenting cells to CD4 ϩ T cells. It is a key enzyme for the degradation of the class II-associated invariant chain, a process that is required for effective antigen loading of class II molecules. Here, we report a selective, orally available, high-affinity cathepsin S inhibitor, 1-, that represents a novel class of immunosuppressive compounds. JNJ 10329670 is a highly potent (K i of ϳ30 nM), nonpeptidic, noncovalent inhibitor of human cathepsin S, but it is much less active against the mouse, dog, monkey, and bovine enzymes. The compound is inactive against other proteases, including the closely related cathepsins L, F, and K. This selectivity makes JNJ 10329670 an excellent tool for exploring the role of cathepsin S in human systems. Treatment of human B cell lines and primary human dendritic cells with JNJ 10329670 resulted in the accumulation of the p10 fragment of the invariant chain (IC 50 of ϳ1 M). In contrast, inhibition of invariant chain proteolysis was much less effective in a human monocytic cell line, suggesting that other enzymes may degrade the invariant chain in this cell type. JNJ 10329670 was shown to block the proteolysis of the invariant chain in vivo by using immunocompromised mice injected with human peripheral blood mononuclear cells (PBMCs). Furthermore, this inhibitor blocks the presentation of tetanus toxoid and giant ragweed by human PBMCs. The properties of JNJ 10329670 make it a candidate for immunosuppressive therapy of allergies and autoimmune diseases. The presentation of antigens by MHC class II (MHC II) molecules is the crucial initiating step in a CD4ϩ T cellmediated immune response. Antigen presenting cells, mainly dendritic cells, B cells, and macrophages, take up and present antigens from the extracellular environment. The internalized protein antigens are processed by endosomal or lysosomal proteases to generate peptides that become associated with MHC II molecules. Peptide-loaded MHC II molecules are subsequently transported to the cell surface for display to CD4 ϩ T cells. Recognition of the MHC II/peptide complexes triggers the activation of antigen-specific CD4 ϩ T cells, which in turn activate other components of the immune system such as B cells, macrophages, and CD8 ϩ T cells. These cellular responses are crucial for the body's response to pathogens, but they are also responsible for the development and symptoms of allergy and autoimmune disease.Cathepsin S is a cysteine protease found in the lysosome of hematopoietic cells. It is a member of the papain superfamily and has 57% identity to cathepsins L and K. Unlike most other lysosomal proteases that are only active under acidic conditions, the activity of cathepsin S exhibits a broad pH optimum that extends to alkaline pH. In contrast to the housekeeping enzymes cathepsins B, D, and L, which are expressed ubiquitously, cathepsin S is expressed mainly in dendritic cells,...

ABSTRACTpretreatment with tipifarnib resulted in significant inhibition of TNF-␣, IL-6, MCP-1, IL-1␤, and MIP-1␣ production. Tipifarnib had no effect in vitro or in vivo on LPS-induced IL-8. Studies in THP-1 cells to address potential mechanism(s) showed that tipifarnib partially inhibited LPS-induced p38 phosphorylation. Tipifarnib significantly inhibited inhibitory subunit of nuclear factor-B (NF-B) (IB)-␣ degradation and p65 nuclear translocation induced by LPS, but not by tumor necrosis factor-␣, IL-1␣, or toll-like receptor (TLR)2 ligand, suggesting that the target for inhibition of NF-B activation was exclusive to the LPS/TLR4 signal pathway. The extent of IB-␣ degradation inhibition did not correlate with inhibition of Ras farnesylation, indicating that Ras was not the target for the observed antiinflammatory activity of tipifarnib. Our findings differ from those for other FTIs, which may have relevance for their dissimilar activity in specific tumor repertoires.The links between cancer and inflammation are well established (for review, see Balkwill et al., 2005). Many human and murine cancers are found in a microenvironment rich in cytokines, chemokines, and inflammatory enzymes. It is therefore of interest to investigate the effects of antitumor drugs on inflammation. Tipifarnib [R115777, Zarnestra, (R)-6-amino[(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl2(1H)-quinolinone)] is an orally active inhibitor of protein farnesyltransferase. It has shown significant antitumor activity and is currently in clinical trials for acute myeloid leukemia and myelodysplastic syndrome (Cortes, 2003). Farnesyltransferase is an enzyme that catalyzes the attachment of a farnesyl group, from farnesyl pyrophosphate, to the cysteine-thiol group of protein C-terminal CAAX consensus sequences (Moores et al., 1991;Reiss et al., 1991). A variety of cellular proteins are farnesylated (Schafer and Rine, 1992;Tamanoi et al., 2001), including Ras superfamily G proteins, nuclear lamins A and B, rhodopsin kinase, centromere-binding proteins CENP-E and CENP-F, cochaperone DnaJ/HDJ-2, prostacyclin receptor Kinsella, 2004, 2005), and cytosolic phospholipase A 2 ␥ (Jenkins et al., 2003). Ras farnesylation is critical for oncogenic Ras signaling (Kato et al., 1992), and Ras mutants are associated with ϳ30% of human cancers. Farnesyltransferase inhibitors (FTIs) were developed to Article, publication date, and citation information can be found at

The chemokine receptor CXCR4 mediates cell anchorage in the bone marrow (BM) microenvironment and is overexpressed in 25–30% of patients with acute myeloid leukemia (AML). Here we have shown that a new CXCR4 receptor antagonist IgG1 antibody (PF-06747143) binds strongly to AML cell lines and to AML primary cells inhibiting their chemotaxis in response to CXCL12. PF-06747143 also induced cytotoxicity in AML cells via Fc-effector function. To characterize the effects of PF-06747143 on leukemia progression, we used two different patient-derived xenograft (PDX) models: Patient 17CXCR4-low and P15CXCR4-high models, characterized by relatively low and high CXCR4 expression, respectively. Weekly administration of PF-06747143 to leukemic mice significantly reduced leukemia development in both models. Secondary transplantation of BM cells from PF-06747143-treated or IgG1 control-treated animals showed that leukemic progenitors were also targeted by PF-06747143. Administration of a single dose of PF-06747143 to PDX models induced rapid malignant cell mobilization into the peripheral blood (PB). These findings support evaluation of this antibody in AML therapy, with particular appeal to patients resistant to chemotherapy and to unfit patients, unable to tolerate intensive chemotherapy.