Targeting Bruton's tyrosine kinase in B cell malignancies.

Bruton's tyrosine kinase (BTK) is a key component of B cell receptor (BCR) signalling and functions as an important regulator of cell proliferation and cell survival in various B cell malignancies. Small-molecule inhibitors of BTK have shown antitumour activity in animal models and, recently, in clinical studies. High response rates were reported in patients with chronic lymphocytic leukaemia and mantle cell lymphoma. Remarkably, BTK inhibitors have molecular effects that cannot be explained by the classic role of BTK in BCR signalling. In this Review, we highlight the importance of BTK in various signalling pathways in the context of its therapeutic inhibition.

The ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour in humans and is highly resistant to current treatment modalities. We have explored the combined treatment of the endoplasmic reticulum (ER) stress-inducing agent 2,5-dimethyl-celecoxib (DMC) and TNF-related apoptosis-inducing ligand (TRAIL WT) or the DR5-specific TRAIL D269H/E195R variant as a potential new strategy to eradicate GBM cells using TRAIL-resistant and -sensitive GBM cells. GBM cell lines were investigated for their sensitivity to TRAIL, DMC and combination of both agents. Cell viability was measured by MTS assay and apoptosis was assessed by Annexin V/PI and acridine orange staining. Caspase activation and protein expression levels were analysed with Western blotting. Death Receptor (DR) cell surface expression levels were quantified by flow cytometry. DR5 expression was increased in U87 cells by ectopic expression using a retroviral plasmid and survivin expression was silenced using specific siRNAs. We demonstrate that A172 expresses mainly DR5 on the cell surface and that these cells show increased sensitivity for the DR5-specific rhTRAIL D269H/E195R variant. In contrast, U87 cells show low DR cell surface levels and is insensitive via both DR4 and DR5. We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells. The dramatic decrease in cell viability is not accompanied by a correspondent increase in Annexin V/PI or caspase activation typically seen in apoptotic or/and necrotic cells within 24h of treatment. Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells. In A172 cells, sub-toxic concentrations of DMC greatly potentiated TRAIL-induced apoptosis. Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis. Our findings corroborate that DMC is a promising agent against GBM, and uncovers a potential synergistic cooperation with TRAIL in this highly malignant cancer.

Erratum to: the ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma.

[This corrects the article DOI: 10.1186/2193-1801-3-495.].

MAPK p38 and JNK have opposing activities on TRAIL-induced apoptosis activation in NSCLC H460 cells that involves RIP1 and caspase-8 and is mediated by Mcl-1.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce both caspase-dependent apoptosis and kinase activation in tumor cells. Here, we examined the consequences and mechanisms of TRAIL-induced MAPKs p38 and JNK in non-small cell lung cancer (NSCLC) cells. In apoptosis sensitive H460 cells, these kinases were phosphorylated, but not in resistant A549 cells. Time course experiments in H460 cells showed that induction of p38 phosphorylation preceded that of JNK. To explore the function of these kinases in apoptosis activation by TRAIL, chemical inhibitors or siRNAs were employed to impair JNK or p38 functioning. JNK activation counteracted TRAIL-induced apoptosis whereas activation of p38 stimulated apoptosis. Notably, the serine/threonine kinase RIP1 was cleaved following TRAIL treatment, concomitant with detectable JNK phosphorylation. Further examination of the role of RIP1 by short hairpin (sh)RNA-dependent knockdown or inhibition by necrostatin-1 showed that p38 can be phosphorylated in both RIP1-dependent and -independent manner, whereas JNK phosphorylation occurred independent of RIP1. On the other hand JNK appeared to suppress RIP1 cleavage via an unknown mechanism. In addition, only the activation of JNK by TRAIL was caspase-8-dependent. Finally, we identified Mcl-1, a known substrate for p38 and JNK, as a downstream modulator of JNK or p38 activity. Collectively, our data suggest in a subset of NSCLC cells a model in which TRAIL-induced activation of p38 and JNK have counteracting effects on Mcl-1 expression leading to pro- or anti-apoptotic effects, respectively. Strategies aiming to stimulate p38 and inhibit JNK may have benefit for TRAIL-based therapies in NSCLC.

Bruton's tyrosine kinase mediated signaling enhances leukemogenesis in a mouse model for chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL) signals from the B cell receptor (BCR) play a major role in disease development and progression. In this light, new therapies that specifically target signaling molecules downstream of the BCR continue to be developed. While first studies on the selective small molecule inhibitor of Bruton's tyrosine kinase (Btk), Ibrutinib (PCI-32765), demonstrated that Btk inhibition sensitizes CLL cells to apoptosis and alters their migratory behavior, these studies however did not address whether Btk-mediated signaling is involved in the process of CLL leukemogenesis. To investigate the requirement of Btk signaling for CLL development, we modulated Btk expression in the IgH.ETµ CLL mouse model, which is based on sporadic expression of the simian oncovirus SV40 T-antigen in mature B cells. To this end, we crossed IgH.ETµ mice on a Btk-deficient background or introduced a human Btk transgene (CD19-hBtk). Here we show that Btk deficiency fully abrogates CLL formation in IgH.ETµ mice, and that leukemias formed in Btk haplo-insufficient mice selectively expressed the wild-type Btk allele on their active X chromosome. Conversely, Btk overexpression accelerated CLL onset, increased mortality, and was associated with selection of non-stereotypical BCRs into CLL clones. Taken together, these data show that Btk expression represents an absolute prerequisite for CLL development and that Btk mediated signaling enhances leukemogenesis in mice. We therefore conclude that in CLL Btk expression levels set the threshold for malignant transformation.

Kinome profiling of non-canonical TRAIL signaling reveals RIP1-Src-STAT3-dependent invasion in resistant non-small cell lung cancer cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) triggers apoptosis selectively in tumor cells through interaction with TRAIL-R1/DR4 or TRAIL-R2/DR5 and this process is considered a promising avenue for cancer treatment. TRAIL resistance, however, is frequently encountered and hampers anti-cancer activity. Here we show that whereas H460 non-small cell lung cancer (NSCLC) cells display canonical TRAIL-dependent apoptosis, A549 and SW1573 NSCLC cells are TRAIL resistant and display pro-tumorigenic activity, in particular invasion, following TRAIL treatment. We exploit this situation to contrast TRAIL effects on the kinome of apoptosis-sensitive cells to that of NSCLC cells in which non-canonical effects predominate, employing peptide arrays displaying 1024 different kinase pseudosubstrates more or less comprehensively covering the human kinome. We observed that failure of a therapeutic response to TRAIL coincides with the activation of a non-canonical TRAIL-induced signaling pathway involving, amongst others, Src, STAT3, FAK, ERK and Akt. The use of selective TRAIL variants against TRAIL-R1 or TRAIL-R2 subsequently showed that this non-canonical migration and invasion is mediated through TRAIL-R2. Short-hairpin-mediated silencing of RIP1 kinase prevented TRAIL-induced Src and STAT3 phosphorylation and reduced TRAIL-induced migration and invasion of A549 cells. Inhibition of Src or STAT3 by shRNA or chemical inhibitors including dasatinib and 5,15-diphenylporphyrin blocked TRAIL-induced invasion. FAK, AKT and ERK were activated in a RIP1-independent way and inhibition of AKT sensitized A549 cells to TRAIL-induced apoptosis. We thus identified RIP1-dependent and -independent non-canonical TRAIL kinase cascades in which Src and AKT are instrumental and could be exploited as co-targets in TRAIL therapy for NSCLC.

The role of B cell receptor stimulation in CLL pathogenesis.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults in the Western world and is characterized by the accumulation of monoclonal CD5(+) mature B cells in the blood. The disease has a highly variable clinical course. CLL is subdivided into two disease subtypes, whereby leukemias with hypermutated immunoglobulin heavy chain variable (IGHV) genes have a more favorable prognosis than those with unmutated IGHV genes, which tend to show advanced, progressive disease, adverse cytogenetic features and resistance to therapy. The current view is that both CLL types derive from antigen-experienced cells. Based on the finding that the IGHV repertoire is highly restricted and biased, as compared to the normal adult B-cell repertoire, it has been hypothesized that CLL cells are selected by some sort of antigenic pressure. Hereby, either autoantigens or antigens derived from apoptotic cells or pathogens are essential to trigger CLL pathogenesis. Although different cytogenetic aberrations were shown to contribute to CLL leukemogenesis, it remains unclear which abnormalities are primary events. Very recently, whole-genome sequencing identified genes that are recurrently mutated and provided novel insights into the mechanisms of oncogenic transformation. Because of the impact on prognosis, it is important to unravel the role of antigenic selection in CLL. Interestingly, B cell receptor (BCR) signaling is aberrantly increased in CLL and expression of tyrosine kinase ZAP70, which is able to signal downstream of the BCR, is a prognostic indicator. In this context we discuss the functional significance of antigenic selection in CLL and describe emerging agents to target BCR signaling that are currently being tested as a novel therapeutic strategy for CLL.

Persistence of immunoglobulin-producing cells in parotid salivary glands of patients with primary Sjögren's syndrome after B cell depletion therapy.

OBJECTIVES: To assess the persistence of immunoglobulin-producing cell populations in the parotid salivary glands of patients with primary Sjögren's syndrome (pSS) after B cell depletion therapy with rituximab. METHODS: Thirteen patients with pSS and four control patients were included in this study. Patients with pSS were treated with rituximab or placebo. Sequence analysis was carried out on IgA- and IgG-encoding transcripts extracted from parotid salivary gland biopsy specimens taken before treatment and at 12-16 and 36-52 weeks after treatment. RESULTS: At baseline, many clonally related sequences were seen in patients with pSS. The number of clonal expansions was significantly higher in patients with pSS than in control patients. Clonal expansions were composed of IgA- and/or IgG-expressing cells. Rituximab did not significantly alter the degree of clonal expansions. Groups of clonally related cells had members which were shared between biopsy specimens taken before and after treatment. Mutation frequencies of immunoglobulin sequences from clonally related cells in patients with pSS were higher after treatment. CONCLUSIONS: Rituximab treatment does not alter the characteristic features of increased clonal expansions seen in the parotid salivary glands of patients with pSS. The presence of clonally related immunoglobulin-producing cells before and after rituximab treatment strongly suggests that immunoglobulin-producing cells persist in salivary glands of patients with pSS despite B cell depletion. The presence of mixed isotype expression within groups of clonally related cells indicates local class switching in salivary glands of patients with pSS. Persistent immunoglobulin-producing cells may underlie disease relapse after treatment.

E. coli-produced BMP-2 as a chemopreventive strategy for colon cancer: a proof-of-concept study.

Colon cancer is a serious health problem, and novel preventive and therapeutical avenues are urgently called for. Delivery of proteins with anticancer activity through genetically modified bacteria provides an interesting, potentially specific, economic and effective approach here. Interestingly, bone morphogenetic protein 2 (BMP-2) is an important and powerful tumour suppressor in the colon and is thus an attractive candidate protein for delivery through genetically modified bacteria. It has not been shown, however, that BMP production in the bacterial context is effective on colon cancer cells. Here we demonstrate that transforming E. coli with a cDNA encoding an ileal-derived mature human BMP-2 induces effective apoptosis in an in vitro model system for colorectal cancer, whereas the maternal organism was not effective in this respect. Furthermore, these effects were sensitive to cotreatment with the BMP inhibitor Noggin. We propose that prevention and treatment of colorectal cancer using transgenic bacteria is feasible.

Evidence for local expansion of IgA plasma cell precursors in human ileum.

IgA plays a crucial role in establishment and maintenance of mucosal homeostasis between host cells and commensal bacteria. To this end, numerous IgA plasma cells are located in the intestinal lamina propria. Whether the (immediate) precursor cells for these plasma cells can expand locally is not completely known and was studied here. The total number of IgA plasma cells in human ileal biopsies was counted. Sequence analysis of IgA V(H) genes from human ileal biopsies revealed the occurrence of many clonally related sequences within a biopsy, but not between different biopsies. This observation strongly argues for local expansion of IgA precursor cells. By comparing the number of unique sequences with the number of clonally related sequences within a biopsy, we estimated that approximately 100-300 precursors were responsible for the 75,000 IgA-producing cells that were present per biopsy. These precursor cells must therefore have divided locally 9-10 times. Since all sequences contained mutations and most of the mutations present in clonally related sequences were shared, the IgA precursor cells must have arrived initially as mutated cells in the lamina propria. Our data show evidence for the existence of two waves of expansion for IgA-producing cells in human ileum. The first wave occurs during initial stimulation in germinal centers as evidenced by somatic hypermutations. A second wave of expansion of IgA-committed cells occurs locally within the lamina propria as evidenced by the high frequency of clonally related cells.

Modulation of Src activity by low molecular weight protein tyrosine phosphatase during osteoblast differentiation.

BACKGROUND: Src kinase plays a critical role in bone metabolism, particularly in osteoclasts. However, the ability of Src kinase to modulate the activity of other bone cells is less well understood. In this work, we examined the expression and activity of Src and low molecular weight protein tyrosine phosphatase (LMWPTP) during osteoblast differentiation and assessed the modulation of Src kinase by LMWPTP. METHODS: Differentiation of MC3T3-E1 pre-osteoblasts was induced by incubation with ascorbic acid and beta-glycerophosphate for up to 28 days. Src phosphorylation and LMWPTP expression were analyzed by immunoblotting. Src dephosphorylation in vitro was assessed by incubating immunoprecipitated Src with LMWPTP followed by assay of the residual Src activity using Sam68 as substrate. The importance of LMWPTP in Src dephosphorylation was confirmed by silencing pre-osteoblasts with siRNA-LMWPTP and then assessing Src phosphorylation. RESULTS: Pre-osteoblast differentiation was accompanied by a decrease in phosphorylation of the activator site of Src and an increase in phosphorylation of the inhibitory site. The expression of total Src was unaltered, indicating that post-translational modifications play a pivotal role in Src function. LMWPTP expression was higher in periods when the activator site of Src was dephosphorylated. LMWPTP dephosphorylated pY(527)-Src and pY(416)-Src in vitro, with greater specificity for pY(527)Src. Activation of LMWPTP produced strong activation of Src mediated by fast dephosphorylation of pY(527)-Src, followed by slower deactivation of this kinase via dephosphorylation of pY(416)Src. CONCLUSION: These results provide new insight into the mechanisms governing the dynamics of Src activity during osteoblast differentiation. A fuller understanding of these mechanisms will improve our knowledge of bone metabolism and of the regulation of Src in other types of cells.

Human scFv SIgA expressed on Lactococcus lactis as a vector for the treatment of mucosal disease.

The gastrointestinal tract is a complex niche and the main port of entry of many pathogens that trigger a wide range of diseases like inflammatory bowel disease (IBD) and colon cancer. Antibodies are effective for treating such diseases, but a system capable of local delivery at the site of the pathology, thus avoiding systemic side effects, is not yet available. Here we report a novel recombinant scFvSIgA1 protein produced by Lactococcus lactis, anchored to the bacterial membrane, which retains its full immuno-recognizing potential. This scFv fragment employed was specific for a colon cancer epitope, epithelial glycoprotein protein-2 (EGP-2). Accordingly L. lactis expressing this chimeric protein was capable of binding cells expressing this epitope. Expression of specific antibodies on bacteria may allow local delivery of anticancer agents produced by such bacteria in conjunction with the antibody and provides a new avenue in the quest for targeted drug delivery.

Transgenic probiotica as drug delivery systems: the golden bullet?

Functional human proteins are constitutively produced in genetically modified bacteria that survive on human mucosal surfaces, to the benefit of the host. The successful Phase I clinical trial with IL-10-producing Lactococcus lactis for Crohn's disease has opened new avenues for the use of transgenic bacteria as delivery vehicles. The major advantage of this novel strategy is the avoidance of systemic side effects associated with conventional therapies. This methodology opens up an alternative method for local delivery of therapeutic proteins to various mucosal tissues.