The pan phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) blocks survival, adhesion and proliferation of primary chronic lymphocytic leukemia cells.

The phosphoinositide 3-kinases (PI3Ks) are critical components of the B-cell receptor (BCR) pathway and have an important role in the pathobiology of chronic lymphocytic leukemia (CLL). Inhibitors of PI3Kδ block BCR-mediated cross-talk between CLL cells and the lymph node microenvironment and provide significant clinical benefit to CLL patients. However, the PI3Kδ inhibitors applied thus far have limited direct impact on leukemia cell survival and thus are unlikely to eradicate the disease. The use of inhibitors of multiple isoforms of PI3K might lead to deeper remissions. Here we demonstrate that the pan-PI3K/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) was more pro-apoptotic to CLL cells--irrespective of their ATM/p53 status--than PI3Kα or PI3Kδ isoform selective inhibitors. Furthermore, SAR245409 blocked CLL survival, adhesion and proliferation. Moreover, SAR245409 was a more potent inhibitor of T-cell-mediated production of cytokines, which support CLL survival. Taken together, our in vitro data provide a rationale for the evaluation of a pan-PI3K inhibitor in CLL patients.

Structure of Arp2/3 complex in its activated state and in actin filament branch junctions.

The seven-subunit Arp2/3 complex choreographs the formation of branched actin networks at the leading edge of migrating cells. When activated by Wiskott-Aldrich Syndrome protein (WASp), the Arp2/3 complex initiates actin filament branches from the sides of existing filaments. Electron cryomicroscopy and three-dimensional reconstruction of Acanthamoeba castellanii and Saccharomyces cerevisiae Arp2/3 complexes bound to the WASp carboxy-terminal domain reveal asymmetric, oblate ellipsoids. Image analysis of actin branches indicates that the complex binds the side of the mother filament, and Arp2 and Arp3 (for actin-related protein) are the first two subunits of the daughter filament. Comparison to the actin-free, WASp-activated complexes suggests that branch initiation involves large-scale structural rearrangements within Arp2/3.

Activation of Arp2/3 complex-mediated actin polymerization by cortactin.

Cortactin, a filamentous actin (F-actin)-associated protein and prominent substrate of Src, is implicated in progression of breast tumours through gene amplification at chromosome 11q13. However, the function of cortactin remains obscure. Here we show that cortactin co-localizes with the Arp2/3 complex, a de novo actin nucleator, at dynamic particulate structures enriched with actin filaments. Cortactin binds directly to the Arp2/3 complex and activates it to promote nucleation of actin filaments. The interaction of cortactin with the Arp2/3 complex occurs at an amino-terminal domain that is rich in acidic amino acids. Mutations in a conserved amino-acid sequence of DDW abolish both the interaction with the Arp2/3 complex and complex activation. The N-terminal domain is not only essential but also sufficient to target cortactin to actin-enriched patches within cells. Interestingly, the ability of cortactin to activate the Arp2/3 complex depends on an activity for F-actin binding, which is almost 20-fold higher than that of the Arp2/3 complex. Our data indicate a new mechanism for activation of actin polymerization involving an enhanced interaction between the Arp2/3 complex and actin filaments.

Diversion of cytoskeletal processes by Shigella during invasion of epithelial cells.

Shigella, the causative agent of bacillar dysentery, invades colonic epithelial cells and moves intracellularly to spread from cell to cell. The processes of Shigella entry, determined by the Ipa proteins, and of actin-based motility, dependent on the IcsA/VirG protein, represent different levels of bacterial manipulation of the cell cytoskeleton.

GRB2 links signaling to actin assembly by enhancing interaction of neural Wiskott-Aldrich syndrome protein (N-WASp) with actin-related protein (ARP2/3) complex.

Proteins of the Wiskott-Aldrich Syndrome protein (WASp) family connect signaling pathways to the actin polymerization-driven cell motility. The ubiquitous homolog of WASp, N-WASp, is a multidomain protein that interacts with the Arp2/3 complex and G-actin via its C-terminal WA domain to stimulate actin polymerization. The activity of N-WASp is enhanced by the binding of effectors like Cdc42-guanosine 5'-3-O-(thio)triphosphate, phosphatidylinositol bisphosphate, or the Shigella IcsA protein. Here we show that the SH3-SH2-SH3 adaptor Grb2 is another activator of N-WASp that stimulates actin polymerization by increasing the amount of N-WASp. Arp2/3 complex. The concentration dependence of N-WASp activity, sedimentation velocity and cross-linking experiments together suggest that N-WASp is subject to self-association, and Grb2 enhances N-WASp activity by binding preferentially to its active monomeric form. Use of peptide inhibitors, mutated Grb2, and isolated SH3 domains demonstrate that the effect of Grb2 is mediated by the interaction of its C-terminal SH3 domain with the proline-rich region of N-WASp. Cdc42 and Grb2 bind simultaneously to N-WASp and enhance actin polymerization synergistically. Grb2 shortens the delay preceding the onset of Escherichia coli (IcsA) actin-based reconstituted movement. These results suggest that Grb2 may activate Arp2/3 complex-mediated actin polymerization downstream from the receptor tyrosine kinase signaling pathway.

Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility.

To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration at the bacterial surface. On the other hand, the NH(2)-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.

Control of actin filament length and turnover by actin depolymerizing factor (ADF/cofilin) in the presence of capping proteins and ARP2/3 complex.

The effect of Arabidopsis thaliana ADF1 and human ADF on the number of filaments in F-actin solutions has been examined using a seeded polymerization assay. ADF did not sever filaments in a catalytic fashion, but decreased the steady-state length distribution of actin filaments in correlation with its effect on actin dynamics. The increase in filament number was modest as compared with the large increase in filament turnover. ADF did not decrease the length of filaments shorter than 1 micrometer. ADF promoted the rapid turnover of gelsolin-capped filaments in a manner dependent on the number of pointed ends. To explain these results, we propose that, as a consequence of the cooperative binding of ADF to F-actin, two populations of energetically different filaments coexist in solution pending a flux of subunits from one to the other. The ADF-decorated filaments depolymerize rapidly from their pointed ends, while undecorated filaments polymerize. ADF also promotes rapid turnover of gelsolin-capped filaments in the presence of the pointed end capper Arp2/3 complex. It is shown that the Arp2/3 complex steadily generates new barbed ends in solutions of gelsolin-capped filaments, which represents an important aspect of its function in actin-based motility.

Rho family GTPases control entry of Shigella flexneri into epithelial cells but not intracellular motility.

Shigella flexneri, an invasive bacterial pathogen, promotes formation of two cytoskeletal structures: the entry focus that mediates bacterial uptake into epithelial cells and the actin-comet tail that enables the bacteria to spread intracellularly. During the entry step, secretion of bacterial invasins causes a massive burst of subcortical actin polymerization leading the formation of localised membrane projections. Fusion of these membrane ruffles leads to bacterial internalization. Inside the cytoplasm, polar expression of the IcsA protein on the bacterial surface allows polymerization of actin filaments and their organization into an actin-comet tail leading to bacterial spread. The Rho family of small GTPases plays an essential role in the organization and regulation of cellular cytoskeletal structures (i.e. filopodia, lamellipodia, adherence plaques and intercellular junctions). We show here that induction of Shigella entry foci is controlled by the Cdc42, Rac and Rho GTPases, but not by RhoG. In contrast, actin-driven intracellular motility of Shigella does not require Rho GTPases. Therefore, Shigella appears to manipulate the epithelial cell cytoskeleton both by Rho GTPase-dependent and -independent processes.

A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii.

Listeria monocytogenes, Shigella flexneri, and Rickettsia conorii are three bacterial pathogens that are able to polymerize actin into 'comet tail' structures and move within the cytosol of infected cells. The actin-based motilities of L. monocytogenes and S. flexneri are known to require the bacterial proteins ActA and IcsA, respectively, and several mammalian cytoskeleton proteins including the Arp2/3 complex and VASP (vasodilator-stimulated phosphoprotein) for L. monocytogenes and vinculin and N-WASP (the neural Wiskott-Aldrich syndrome protein) for S. flexneri. In contrast, little is known about the motility of R. conorii. In the present study, we have analysed the actin-based motility of this bacterium in comparison to that of L. monocytogenes and S. flexneri. Rickettsia moved at least three times more slowly than Listeria and Shigella in both infected cells and Xenopus laevis egg extracts. Decoration of actin with the S1 subfragment of myosin in infected cells showed that the comet tails of Rickettsia have a structure strikingly different from those of L. monocytogenes or S. flexneri. In Listeria and Shigella tails, actin filaments form a branching network while Rickettsia tails display longer and not cross-linked actin filaments. Immunofluorescence studies revealed that the two host proteins, VASP and (&agr;)-actinin colocalized with actin in the tails of Rickettsia but neither the Arp2/3 complex which we detected in the Shigella actin tails, nor N-WASP, were detected in Rickettsia actin tails. Taken together, these results suggest that R. conorii may use a different mechanism of actin polymerization.

Rupture of the intestinal epithelial barrier and mucosal invasion by Shigella flexneri.

Invasion of the intestinal barrier by Shigella flexneri involves complex interactions with epithelial and phagocytic cells. Major perturbation of the signals that maintain epithelial integrity permits mucosal invasion, leading to tissue destruction. Expression of this invasive phenotype depends on the secretion of Ipa proteins (invasins), which can trigger entry of the pathogen into epithelial cells by causing massive rearrangement of the host cell cytoskeleton and cause macrophage apoptotic death by direct interaction of IpaB with interleukin-1beta (IL-1beta)-converting enzyme. This results in the killing of defense cells and in the release of IL-1beta. In vivo, bacteria translocate through the epithelial barrier, essentially via M cells of the follicle-associated epithelium in the colonic and rectal mucosa. Apoptotic death of macrophages in subepithelial tissues allows bacterial survival and triggers inflammation, which destabilizes epithelial structures and facilitates further bacterial entry. Once they are intracellular, bacteria multiply within the cytoplasm and move from cell to cell by an actin-dependent process.

Molecular bases of epithelial cell invasion by Shigella flexneri.

The pathogenesis of shigellosis is characterized by the capacity of the causative microorganism, Shigella, to invade the epithelial cells that compose the mucosal surface of the colon in humans. The invasive process encompasses several steps which can be summarized as follows: entry of bacteria into epithelial cells involves signalling pathways that elicit a macropinocitic event. Upon contact with the cell surface, S. flexneri activates a Mxi/Spa secretory apparatus encoded by two operons comprising about 25 genes located on a large virulence plasmid of 220 kb. Through this specialized secretory apparatus, Ipa invasins are secreted, two of which (IpaB, 62 kDa and IpaC, 42 kDa) form a complex which is itself able to activate entry via its interaction with the host cell membrane. Interaction of this molecular complex with the cell surface elicits major rearrangements of the host cell cytoskeleton, essentially the polymerization of actin filaments that form bundles supporting the membrane projections which achieve bacterial entry. Active recruitment of the protooncogene pp 60c-src has been demonstrated at the entry site with consequent phosphorylation of cortactin. Also, the small GTPase Rho is controlling the cascade of signals that allows elongation of actin filaments from initial nucleation foci underneath the cell membrane. The regulatory signals involved as well as the proteins recruited indicate that Shigella induces the formation of an adherence plaque at the cell surface in order to achieve entry. Once intracellular, the bacterium lyses its phagocytic vacuole, escapes into the cytoplasm and starts moving the inducing polar, directed polymerization of actin on its surface, due to the expression of IcsA, a 120 kDa outer membrane protein, which is localized at one pole of the microorganism, following cleavage by SopA, a plasmid-encoded surface protease. In the context of polarized epithelial cells, bacteria then reach the intermediate junction and engage their components, particularly the cadherins, to form a protrusion which is actively internalized by the adjacent cell. Bacteria then lyse the two membranes, reach the cytoplasmic compartment again, and resume actin-driven movement.

SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri.

The spreading ability of Shigella flexneri, a facultative intracellular Gram-negative bacterium, within the host-cell cytoplasm is the result of directional assembly and accumulation of actin filaments at one pole of the bacterium. IcsA/VirG, the 120 kDa outer membrane protein that is required for intracellular motility, is located at the pole of the bacterium where actin polymerization occurs. Bacteria growing in laboratory media and within infected cells release a certain proportion of the surface-exposed IcsA after proteolytic cleavage. In this study, we report the characterization of the sopA gene which is located on the virulence plasmid and encodes the protein responsible for the cleavage of IcsA. The deduced amino acid sequence of SopA exhibits 60% identity with those of the OmpT and OmpP outer membrane proteases of Escherichia coli. The construction and phenotypic characterization of a sopA mutant demonstrated that SopA is required for exclusive polar localization of IcsA on the bacterial surface and proper expression of the motility phenotype in infected cells.

Characterization of a monoclonal IgMK (IgMGAS) anti-Gd cold agglutinin (CA). Its coexistence with a monoclonal IgG3K (IgGGAS) without CA activity that might be clonally related to IgMGAS.

A monoclonal IgMK (IgMGAS) cold agglutinin (CA) of the infrequent anti-Gd specificity, found in a patient with Waldenström macroglobulinemia, has been characterized. IgMGAS uses a VH gene homologous (93.7%) to the reported VH251 germ-line, one of the two functional genes of the VH5 family, with differences in both framework regions and complementary determining regions (CDR). The VL gene is homologous to the reported 15AVK1 germ-line gene, recently described in an anti-i CA, with differences mostly clustered in CDR. In the patient's serum, IgMGAS coexisted with a monoclonal IgG3K (IgGGAS) that lacked CA activity but expressed the private idiotopes found on IgMGAS. Both Ig lacked reactivity with antibodies detecting VH1 or VHIII or VKIII subgroup regions or the VH4-21 gene product that is expressed by anti-I/i CAs. The K chains from both Igs showed the same isoelectrical mobility. Moreover, the k chains from both serum Igs showed the same N-terminal amino acid sequence. This sequence was identical to that predicted by the nucleotide sequence of VK1GAS gene segment, including one discrepancy at position 15 (Ile for Val) with respect to the consensus VK1 subgroup regions. Although these data do not exclude a possible independent clonal origin, they are consistent with the notion that IgGGAS might be clonally related to IgMGAS.

V gene usage by seven hybrids derived from CD5+ B-cell chronic lymphocytic leukemia and displaying autoantibody activity.

We report here the complete heavy and light chain variable region sequences of seven heterohybridomas derived from CD5+ chronic lymphocytic leukemia (CLL) B lymphocytes and displaying natural autoantibody activity. The three hybrids displaying a polyreactive pattern of binding used VH4 family members, ie, the VH4-18 gene in germinal configuration in two cases and a VH4 gene with 90% homology with VH4-21 for the third one. A hybrid expressing anti-Sm activity used a VH3 family member with 95.26% homology with the 30P1 gene. The three hybrids exclusively displaying rheumatoid factor activity expressed VH1 family genes: 51P1 gene for two (in germinal configuration in one, and with 93.2% homology in the other), whereas the third one used the V1-3b gene (98.8% homology). Definitive homology with known germline D segments was found for four of the seven hybrids (DN2 in 3 and DLR4 in 1) and JH use appeared to be random. The three hybrids displaying polyreactive activity expressed V kappa I, V lambda III, and V lambda II genes, all in germinal configuration. Among the three hybrids with rheumatoid factor activity, two used the same V kappa II gene with, respectively, 98% and 96% homology with a gene previously described; the third used a V lambda I gene in germinal configuration. Finally, the clone with anti-Sm activity used a V lambda III gene having 97% homology with a germinal gene. Overall, these results attempt to establish the relationship between frequent self-reactivity observed in CD5+ B-CLL and V gene usage. For VH genes, they confirm overexpression of the 51P1 gene in B-CLL and suggest nonstochastic use of two VH4 genes (4-21 and 4-18). For VL genes, available information is too scarce to lead to firm conclusions.