Comprehensive analysis of T cell leukemia signals reveals heterogeneity in the PI3 kinase-Akt pathway and limitations of PI3 kinase inhibitors as monotherapy.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner

A Transgenic Drosophila Model Demonstrates That the Helicobacter pylori CagA Protein Functions as a Eukaryotic Gab Adaptor

Infection with the human gastric pathogen Helicobacter pylori is associated with a spectrum of diseases including gastritis, peptic ulcers, gastric adenocarcinoma, and gastric mucosa–associated lymphoid tissue lymphoma. The cytotoxin-associated gene A (CagA) protein of H. pylori, which is translocated into host cells via a type IV secretion system, is a major risk factor for disease development. Experiments in gastric tissue culture cells have shown that once translocated, CagA activates the phosphatase SHP-2, which is a component of receptor tyrosine kinase (RTK) pathways whose over-activation is associated with cancer formation. Based on CagA's ability to activate SHP-2, it has been proposed that CagA functions as a prokaryotic mimic of the eukaryotic Grb2-associated binder (Gab) adaptor protein, which normally activates SHP-2. We have developed a transgenic Drosophila model to test this hypothesis by investigating whether CagA can function in a well-characterized Gab-dependent process: the specification of photoreceptors cells in the Drosophila eye. We demonstrate that CagA expression is sufficient to rescue photoreceptor development in the absence of the Drosophila Gab homologue, Daughter of Sevenless (DOS). Furthermore, CagA's ability to promote photoreceptor development requires the SHP-2 phosphatase Corkscrew (CSW). These results provide the first demonstration that CagA functions as a Gab protein within the tissue of an organism and provide insight into CagA's oncogenic potential. Since many translocated bacterial proteins target highly conserved eukaryotic cellular processes, such as the RTK signaling pathway, the transgenic Drosophila model should be of general use for testing the in vivo function of bacterial effector proteins and for identifying the host genes through which they function

Investigating Pathogenic Mechanisms of the Helicobacter pylori Virulence Factor CagA Using Transgenic Expression in Drosophila melanogaster

99 pagesUpon colonization of the human stomach, Helicobacter pylori establishes intimate interactions with the gastric epithelium, resulting in pathogenic host responses that can lead to gastric cancer. An important component of this interaction is translocation of the CagA effector protein into host cells, where it manipulates several conserved signaling pathways. Experiments in tissue culture cells have shown that CagA activates the phosphatase SHP-2, a component of receptor tyrosine kinase (RTK) pathways whose overactivation is associated with cancer formation. CagA has been proposed to function as a prokaryotic mimic of the eukaryotic Gab adaptor protein, which normally activates SHP-2. We developed a transgenic Drosophila melanogaster model to investigate whether CagA can function in a Gab-dependent process: specification of photoreceptor cells in the eye. We demonstrate that CagA expression is sufficient to rescue photoreceptor development in the absence of the Gab homologue through a mechanism that requires Drosophila SHP-2, demonstrating that CagA functions as a Gab protein in vivo and providing insight into CagA’s oncogenic potential. In addition to its function in RTK signaling, we explore CagA’s interactions with other host cell signaling pathways using the transgenic Drosophila model. We show that expressing CagA in the simple model epithelium created during wing development triggers apoptosis through activation of JNK signaling. We demonstrate that loss of several upstream JNK pathway components, including neoplastic tumor suppressors and the homolog of tumor necrosis factor, enhances CagA-induced cell death. Using a Drosophila model of metastasis we show that CagA enhances growth and invasion of tumors generated by expression of oncogenic Ras through JNK activation, implicating this pathway as an important driver of human gastric cancer progression. Finally, we use our transgenic Drosophila model to examine a role for CagA in disrupting the gastrointestinal ecosystem. We show that expressing CagA in adult intestinal stem cells is sufficient to significantly enhance epithelial proliferation, increase the production of antimicrobial peptides, and alter the intestinal bacterial community. This dissertation includes both previously published and unpublished co-authored material.Committee in charge: Kenneth E. Prehoda, Chair; Karen J. Guillemin, Advisor; Christopher A. Doe, Member; Tom H. Stevens, Member; Victoria G. Herman, Outside Member, Kimberly Andrews Espy, Membe

Transgenic Expression of the <em>Helicobacter pylori</em> Virulence Factor CagA Promotes Apoptosis or Tumorigenesis through JNK Activation in <em>Drosophila</em>

<div><p>Gastric cancer development is strongly correlated with infection by <em>Helicobacter pylori</em> possessing the effector protein CagA. Using a transgenic <em>Drosophila melanogaster</em> model, we show that CagA expression in the simple model epithelium of the larval wing imaginal disc causes dramatic tissue perturbations and apoptosis when CagA-expressing and non-expressing cells are juxtaposed. This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells. We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a <em>Drosophila</em> model of metastasis. In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner. These data suggest a potential role for CagA-mediated JNK pathway activation in promoting gastric cancer progression.</p> </div

CagA expression causes apoptosis and epithelial disruption.

<p>(A) Schematic illustrating the fate of different domains within the wing imaginal disc. Each color-coded region of the larval structure on the left corresponds to the specified region of the adult wing on the right (modified from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002939#ppat.1002939-Bate1" target="_blank">[24]</a>). (B–G) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression and stained with an antibody against active caspase-3 to mark apoptotic cells. A control wing disc epithelium expressing only mGFP with the bx-GAL4 dorsal wing driver (B) lacks apoptotic cells. Ubiquitous expression of CagA in the wing disc with the 765-GAL4 driver (C) does not cause apoptosis, while expressing CagA with bx-GAL4 (D) triggers formation of apoptotic clusters within the expression domain. Expressing two copies of CagA with bx-GAL4 (E) causes a dose-dependent enhancement of the apoptosis phenotype. Expressing CagA^EPISA with bx-GAL4 (F) does not cause a phenotype, while expressing two copies of CagA^EPISA (G) produces small apoptotic clusters. Scale bars, 50 µm. (H) XZ confocal plane of a male wing imaginal disc epithelium expressing mGFP and CagA with bx-GAL4 stained with antibodies against active caspase-3 to show basal extrusion of apoptotic cells and matrix metalloproteinase 1 (Mmp1) to show evidence of basement membrane breakdown. Scale bar, 20 µm. (I–N) Adult wing images from male flies of each indicated genotype. Neither expression of mGFP alone with bx-GAL4 (I) nor expression of CagA with 765-GAL4 (J) causes a phenotype in the adult wing. Dorsal wing expression of CagA with bx-GAL4 (K) disrupts epithelial integrity in a dose-dependent manner (L). Expressing CagA^EPISA with bx-GAL4 (M) does not cause an adult wing phenotype, while expressing two copies of CagA^EPISA (N) causes epithelial disruption. Scale bar, 500 µm.</p

CagA enhances tumor invasion through JNK activation.

<p>(A–E) Confocal cross sections of cephalic complexes from third instar larvae with GFP-marked tumors stained with an antibody against ElaV to mark terminally differentiated cells and phalloidin to reveal f-actin structure. VNCs are outlined in panels showing GFP expression, and arrows highlight invading tumor tissue. Expressing Ras^V12 alone in whole eye clones (A) causes a mild invasive phenotype characterized by either no invasion or migration of tumor cells from one optic lobe. Coexpression of CagA with Ras^V12 (B) dramatically enhances the extent of VNC invasion from both optic lobes, while coexpression of CagA^EPISA with Ras^V12 (C) shows a milder enhancement of invasion. Coexpression of Bsk^DN with Ras^V12 (D) does not significantly affect the invasive capacity of tumor cells, while coexpression of Bsk^DN with Ras^V12 and CagA (E) suppresses the VNC invasion phenotype. Scale bar, 50 µm. (F) Projections of several confocal cross sections from third instar larval cephalic complexes with GFP-marked tumors showing different classes of invasiveness: (0) noninvasive, (1) invasion from one optic lobe, (2) invasion from both optic lobes, (3) significant invasion of the VNC. Brain lobes and ventral nerve cords are outlined. Scale bar, 50 µm. (G) Quantitation of the percentage of cephalic complexes classified into each category. The number of samples analyzed is shown above each column. * indicates a distribution that differs significantly compared to Ras^V12; † indicates a distribution that differs significantly compared to Ras^V12, CagA; p<0.0001.</p

CagA-induced apoptosis occurs through JNK pathway activation.

<p>(A–E) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression with bx-GAL4 and stained with an anti-active caspase-3 antibody to mark apoptotic cells. Ectopic overexpression of wild type Bsk in the dorsal wing disc (A) causes a mild apoptosis phenotype that is strongly enhanced by coexpression with CagA (B). Coexpression of Bsk with CagA^EPISA (C) also enhances the apoptosis phenotype. Expression of Bsk^DN alone (D) does not cause apoptosis, and coexpression with CagA (E) strongly suppresses apoptosis induced by CagA expression. Scale bars, 50 µm. (F–J) Adult wing images from male flies expressing different forms of Bsk alone or in combination with CagA. Ectopic overexpression of Bsk with bx-GAL4 (F) causes only subtle vein defects in the adult wing, while coexpression with CagA (G) enhances epithelial disruption. Coexpression of Bsk with CagA^EPISA (H) does not significantly affect formation of the adult wing structure. Expression of Bsk^DN with bx-GAL4 (I) also causes only subtle vein defects in the adult wing, while coexpression with CagA (J) enhances epithelial disruption. Arrowheads highlight ectopic veins in adult wings expressing different forms of Bsk alone. Scale bar, 500 µm. (K) Quantitation of apoptosis as a percentage of the expression domain showing active caspase-3 staining, n = 15 wing discs per genotype; bar indicates average value for each group. * indicates values that differ significantly from the control with expression of a single transgene; † indicates values that show significant enhancement or suppression compared to CagA; ‡ indicates values that show significant enhancement compared to CagA^EPISA; p<0.0001. (L) Confocal cross section of a male wing imaginal disc epithelium carrying the <i>puc-lacZ</i> reporter allele and expressing mGFP and CagA with bx-GAL4. Staining with antibodies against active caspase-3 and β-galactosidase (β-gal) shows that apoptotic cells lie adjacent to those in which JNK signaling has been activated. Scale bar, 50 µm. (M) A model of the JNK pathway depicting the multiple upstream activators known to induce JNK-dependent apoptosis in <i>Drosophila</i>, and indicating human homologs for each pathway component.</p

CagA genetically interacts with nTSGs, Eiger and Rho1.

<p>(A–E) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression with bx-GAL4 and stained with anti-active caspase-3 antibody to mark apoptotic cells. Dorsal wing expression of CagA with bx-GAL4 (A) causes formation of apoptotic clusters. RNAi-mediated knockdown of the nTSG Dlg alone (B) does not cause significant apoptosis, but enhances apoptosis induced by CagA expression (C). The apoptosis phenotype is enhanced when CagA is expressed in an <i>egr</i> mutant background (D). Coexpression of Rho1 with CagA (E) also enhances apoptosis. Scale bars, 50 µm. (F) Quantitation of apoptosis as a percentage of the expression domain showing active caspase-3 staining, n = 10 or 15 wing discs per genotype; bar indicates average value for each group. * indicates values that show significant enhancement compared to CagA, whose quantitation (from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002939#ppat-1002939-g002" target="_blank">Figure 2</a>) is provided for comparison; p<0.0001. (G) A model showing the localization of polarity protein complexes in an epithelial cell, their known interactions with other upstream activators of JNK signaling in <i>Drosophila</i>, and the downstream effects of these interactions.</p

Models illustrating short-term effects of CagA on an epithelium and long-term effects resulting from a change in host genetic background.

<p>(A) Once inside the host epithelial cell, CagA effector protein downregulates the neoplastic tumor suppressors (nTSGs) which induces endocytic activation of the TNF homolog Eiger (Egr) leading to activation of JNK (Bsk). CagA also triggers Egr-dependent JNK pathway activation in neighboring wild type cells. In the absence of this pathway, CagA activates JNK signaling through other upstream pathway components including the small GTPase Rho1. In a wild type host genetic background, CagA-mediated JNK pathway activation causes apoptosis and subsequent extrusion from the epithelium, or engulfment by neighboring cells. (B) Introduction of CagA into host cells causes upregulation of JNK signaling which triggers apoptosis and compensatory proliferation within the epithelium as part of the cell editing process. When the host genetic background is perturbed by expression of an oncogenic mutation which blocks apoptosis, CagA-mediated JNK pathway activation drives tumor progression.</p