PI3K in juvenile myelomonocytic leukemia

Indiana University-Purdue University Indianapolis (IUPUI)Juvenile Myelomonocytic Leukemia (JMML) is rare, fatal myeloproliferative disease (MPD) affecting young children, and is characterized by expansion of monocyte lineage cells and hypersensitivity to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) stimulation. JMML is frequently associated with gain-of-function mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase, Shp2. Activating Shp2 mutations are known to promote hyperactivation of the Ras-Erk signaling pathway, but Akt is also observed to have enhanced phosphorylation, suggesting a potential role for Phosphatidylinositol-3-Kinase (PI3K)-Akt signaling in mutant Shp2-induced GM-CSF hypersensitivity and leukemogenesis. Having demonstrated that Class IA PI3K is hyperactivated in the presence of mutant Shp2 and contributes to GM-CSF hypersensitivity, I hypothesized the hematopoietic-specific Class IA PI3K catalytic subunit p110δ is a crucial mediator of mutant Shp2-induced PI3K hyperactivation and GM-CSF hypersensitivity in vitro and MPD development in vivo. I crossed gain-of-function mutant Shp2 D61Y inducible knockin mice, which develop fatal MPD, with mice expressing kinase-dead mutant p110δ D910A to evaluate p110δ’s role in mutant Shp2-induced GM-CSF hypersensitivity in vitro and MPD development in vivo. As a comparison, I also crossed Shp2 D61Y inducible knockin mice with mice bearing inducible knockout of the ubiquitously expressed Class IA PI3K catalytic subunit, p110α. I found that genetic interruption of p110δ, but not p110α, significantly reduced GM-CSF-stimulated hyperactivation of both the Ras-Erk and PI3K-Akt signaling pathways, and as a consequence, resulted in reduced GM-CSF-stimulated hyper-proliferation in vitro. Furthermore, I found that mice bearing genetic disruption of p110δ, but not p110α, in the presence of gain-of-function mutant Shp2 D61Y, had on average, smaller spleen sizes, suggesting that loss of p110δ activity reduced MPD severity in vivo. I also investigated the effects of three PI3K inhibitors with high specificity for p110δ, IC87114, GDC-0941, and GS-9820 (formerly known as CAL-120), on mutant Shp2-induced GM-CSF hypersensitivity. These inhibitors with high specificity for p110δ significantly reduced GM-CSF-stimulated hyperactivation of PI3K-Akt and Ras-Erk signaling and reduced GM-CSF-stimulated hyperproliferation in cells expressing gain-of-function Shp2 mutants. Collectively, these findings show that p110δ-dependent PI3K hyperactivation contributes to mutant Shp2-induced GM-CSF hypersensitivity and MPD development, and that p110δ represents a potential novel therapeutic target for JMML

Reconstructing the depositional history of the Eel River paleo meltwater channel, northeastern Indiana using sediment provenance techniques

Indiana University-Purdue University Indianapolis (IUPUI)The outwash deposits of the Eel River paleo meltwater channel in DeKalb and Allen Counties, Indiana predominantly originated from the Erie Lobe of the Laurentide Ice Sheet, but do contain some sediment from the Saginaw Lobe. This determination helps clarify the ice dynamics and Last Glacial Maximum sediment depostional history in northeastern Indiana, which is complicated because of the interactions between the Erie and Saginaw Lobes. Outwash deposits were analyzed from IGS core SC0802 in the Eel River paleo meltwater channel, which intersects the previously identified Huntertown Formation. The core includes 29.2 m of deposits underlain by the hard glacial till of the Trafalgar formation. Mean grain size, sediment skewness, lithology, magnetic susceptibility, and quantitative X-ray diffraction were used to evaluate the provenance of the outwash deposits. Representative samples of Erie Lobe and Saginaw Lobe deposits were analyzed to develop end member provenance signatures. A weight of evidence approach was developed and revealed that deposits from 8.0-13.8 m are of mixed origin from the Erie and Saginaw Lobes, whereas the 0-8.0 and 13.8-29.2 m deposits are Erie Lobe in origin. Cluster analysis and discriminant function analysis supported the findings of this approach. These findings suggests that the Eel River paleo meltwater channel was formed as an outwash channel, and that the adjacent Huntertown Formation does not appear to have been directly deposited by the Saginaw Lobe. The sediments of Saginaw origin from ~8-14 m in the Eel River paleo meltwater channel were likely transported from an upgradient source. The sediments from this zone have a larger mean grain size indicating deposition occurred during higher meltwater discharge, such as the release of meltwater from the drainage of proglacial or subglacial lake(s) associated with the disintegration of the Saginaw Lobe, thus resulting in the mixing of Saginaw Lobe deposits with Erie Lobe deposits. However, the majority of the sediment in the Eel River paleo channel near SC0802 is Erie Lobe in origin. Based on the provenance and depositional sequence at SC0802, the Saginaw Lobe disintegrated prior to the Erie Lobe retreat from the Wabash moraine around 16-17 cal ka

Genetic disruption of the PI3K regulatory subunits, p85 alpha, p55 alpha, and p50 alpha partially normalizes gain-of-function PTPN11- induced hypersensitivity to GM-CSF in hematopoietic progenitors

poster abstractJuvenile Myelomonocytic Leukemia (JMML) is a lethal myeloproliferative disorder (MPD) of children, characterized by hyperproliferation of myelomonocytic cells and hypersensitivity to Granulocyte-Monocyte Colony-Stimulating Factor (GM-CSF). JMML is frequently associated with gain-of-function mutations in PTPN11, which encodes the protein tyrosine phosphatase, Shp2, and which is known to positively regulate Ras signaling. The role of MAPK signaling in gain-of-function mutant Shp2-induced leukemogenesis is well established. In addition, phosphoAkt levels are elevated in the presence of gain-of-function Shp2 mutations, suggesting a role for Phosphatidyl-Inositol-3-Kinase (PI3K) signaling (Yang, et al, 2008). Class IA PI3K is a lipid kinase heterodimer composed of one of two regulatory subunits—p85 alpha or p85 beta—and one of three catalytic subunits—p110 alpha, p110 beta, or p110 delta. PI3K mediates proliferative and anti-apoptotic signals. We have found that there is increased interaction between the p85 alpha regulatory subunit and the p110 alpha catalytic subunit in gain-offunction mutant Shp2-expressing cells compared to WT Shp2-expressing cells. The p85 alpha regulatory subunit, along with its splice variants, p55 alpha and p50 alpha, is encoded by the gene Pik3r1. To investigate the hypothesis that p85 alpha-dependent PI3K signaling contributes to gain-of-function mutant Shp2-induced GM-CSF hypersensitivity, WT and Pik3r1-/- fetal liver-derived hematopoietic progenitor cells were transduced with WT Shp2 or gain-of-function mutant Shp2 E76K. Ablation of all the Pik3r1 isoforms resulted in a significant, but incomplete, correction of GM-CSF hypersensitivity in Shp2 E76K-expressing cells. Consistently, upon genetic disruption of Pik3r1, Akt phosphorylation was reduced in both WT Shp2- and Shp2 E76K-expressing cells compared to that seen in Pik3r1+/+ cells, but was not completely absent. Additionally, Erk activation was reduced in Pik3r1-/- cells expressing Shp2 E76K compared to that in Pik3r1+/+ cells, indicating that interruption of Shp2-mediated PI3K signaling affects the MAPK pathway as well, which likely contributes to the reduction in GM-CSF-stimulated proliferation in the Pik3r1-/- cells. Finally, treatment with the PI3K inhibitor, LY294002 resulted in complete abrogation of Akt phosphorylation in Pik3r1-/- cells transduced with Shp2 E76K, indicating that residual PI3K activity in the absence of Pik3r1 likely contributes to the incomplete correction of GM-CSF hypersensitivity and suggesting that although p85 alpha plays an important role in gain-of-function mutant Shp2-induced hyperactivation of PI3K signaling, additional p85 alpha-independent mechanisms contribute as well

Report of the Committee on Resolutions- Declaration

Pamphlet concerning a declaration made by the National Educational Association at the forty-fourth annual convention

Electoral bias at the 2015 general election: reducing Labour’s electoral advantage

Electoral bias results in an asymmetrical seat distribution between parties with similar vote shares. Over recent British general elections Labour held an advantage because it efficiently converted votes into seats. Following the 2015 election result this advantage has reduced considerably, principally because Labour’s vote distribution saw it accumulate more ineffective votes, particularly where electoral support was not converted into seats. By contrast, the vote distribution of the Conservative party is now superior to that of Labour because it acquired fewer wasted votes although Labour retains a modest advantage overall because it benefits from inequalities in electorate size and differences in voter turnout. Features of the 2015 election, however, raise general methodological challenges for decomposing electoral bias. The analysis, therefore, considers the effect of substituting the Liberal Democrats as the third party with the United Kingdom Independence Party. It also examines the outcome in Scotland separately from that in England and Wales. Following this analysis it becomes clear that the method for decomposing electoral bias requires clearer guidelines for its application in specific settings

Protein-tyrosine Phosphatase Shp2 Positively Regulates Macrophage Oxidative Burst

Macrophages are vital to innate immunity and express pattern recognition receptors and integrins for the rapid detection of invading pathogens. Stimulation of Dectin-1 and complement receptor 3 (CR3) activates Erk- and Akt-dependent production of reactive oxygen species (ROS). Shp2, a protein-tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt and is crucial for hematopoietic cell function; however, no studies have examined Shp2 function in particulate-stimulated ROS production. Maximal Dectin-1-stimulated ROS production corresponded kinetically to maximal Shp2 and Erk phosphorylation. Bone marrow-derived macrophages (BMMs) from mice with a conditionally deleted allele of Ptpn11 (Shp2flox/flox;Mx1Cre+) produced significantly lower ROS levels compared with control BMMs. Although YFP-tagged phosphatase dead Shp2-C463A was strongly recruited to the early phagosome, its expression inhibited Dectin-1- and CR3-stimulated phospho-Erk and ROS levels, placing Shp2 phosphatase function and Erk activation upstream of ROS production. Further, BMMs expressing gain of function Shp2-D61Y or Shp2-E76K and peritoneal exudate macrophages from Shp2D61Y/+;Mx1Cre+ mice produced significantly elevated levels of Dectin-1- and CR3-stimulated ROS, which was reduced by pharmacologic inhibition of Erk. SIRPα (signal regulatory protein α) is a myeloid inhibitory immunoreceptor that requires tyrosine phosphorylation to exert its inhibitory effect. YFP-Shp2C463A-expressing cells have elevated phospho-SIRPα levels and an increased Shp2-SIRPα interaction compared with YFP-WT Shp2-expressing cells. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated ROS production in macrophages by dephosphorylating and thus mitigating the inhibitory function of SIRPα and by promoting Erk activation

Role of p85α in neutrophil extra- and intracellular reactive oxygen species generation

Drug resistance is a growing problem that necessitates new strategies to combat pathogens. Neutrophil phagocytosis and production of intracellular ROS, in particular, has been shown to cooperate with antibiotics in the killing of microbes. This study tested the hypothesis that p85α, the regulatory subunit of PI3K, regulates production of intracellular ROS. Genetic knockout of p85α in mice caused decreased expression of catalytic subunits p110α, p110β, and p110δ, but did not change expression levels of the NADPH oxidase complex subunits p67phox, p47phox, and p40phox. When p85α, p55α, and p50α (all encoded by Pik3r1) were deleted, there was an increase in intracellular ROS with no change in phagocytosis in response to both Fcγ receptor and complement receptor stimulation. Furthermore, the increased intracellular ROS correlated with significantly improved neutrophil killing of both methicillin-susceptible and methicillin-resistant S. aureus. Our findings suggest inhibition of p85α as novel approach to improving the clearance of resistant pathogens

PI3K p110δ uniquely promotes gain-of-function Shp2-induced GM-CSF hypersensitivity in a model of JMML

Although hyperactivation of the Ras-Erk signaling pathway is known to underlie the pathogenesis of juvenile myelomonocytic leukemia (JMML), a fatal childhood disease, the PI3K-Akt signaling pathway is also dysregulated in this disease. Using genetic models, we demonstrate that inactivation of phosphatidylinositol-3-kinase (PI3K) catalytic subunit p110δ, but not PI3K p110α, corrects gain-of-function (GOF) Shp2-induced granulocyte macrophage-colony-stimulating factor (GM-CSF) hypersensitivity, Akt and Erk hyperactivation, and skewed hematopoietic progenitor distribution. Likewise, potent p110δ-specific inhibitors curtail the proliferation of GOF Shp2-expressing hematopoietic cells and cooperate with mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK) inhibition to reduce proliferation further and maximally block Erk and Akt activation. Furthermore, the PI3K p110δ-specific inhibitor, idelalisib, also demonstrates activity against primary leukemia cells from individuals with JMML. These findings suggest that selective inhibition of the PI3K catalytic subunit p110δ could provide an innovative approach for treatment of JMML, with the potential for limiting toxicity resulting from the hematopoietic-restricted expression of p110δ

A life in progress: motion and emotion in the autobiography of Robert M. La Follette

This article is a study of a La Follette’s Autobiography, the autobiography of the leading Wisconsin progressive Robert M. La Follette, which was published serially in 1911 and, in book form, in 1913. Rather than focusing, as have other historians, on which parts of La Follette’s account are accurate and can therefore be trusted, it explains instead why and how this major autobiography was conceived and written. The article shows that the autobiography was the product of a sustained, complex, and often fraught series of collaborations among La Follette’s family, friends, and political allies, and in the process illuminates the importance of affective ties as well as political ambition and commitment in bringing the project to fruition. In the world of progressive reform, it argues, personal and political experiences were inseparable

Effect of Systemic Iron Overload and a Chelation Therapy in a Mouse Model of the Neurodegenerative Disease Hereditary Ferritinopathy

Mutations in the ferritin light chain (FTL) gene cause the neurodegenerative disease neuroferritinopathy or hereditary ferritinopathy (HF). HF is characterized by a severe movement disorder and by the presence of nuclear and cytoplasmic iron-containing ferritin inclusion bodies (IBs) in glia and neurons throughout the central nervous system (CNS) and in tissues of multiple organ systems. Herein, using primary mouse embryonic fibroblasts from a mouse model of HF, we show significant intracellular accumulation of ferritin and an increase in susceptibility to oxidative damage when cells are exposed to iron. Treatment of the cells with the iron chelator deferiprone (DFP) led to a significant improvement in cell viability and a decrease in iron content. In vivo, iron overload and DFP treatment of the mouse model had remarkable effects on systemic iron homeostasis and ferritin deposition, without significantly affecting CNS pathology. Our study highlights the role of iron in modulating ferritin aggregation in vivo in the disease HF. It also puts emphasis on the potential usefulness of a therapy based on chelators that can target the CNS to remove and redistribute iron and to resolubilize or prevent ferritin aggregation while maintaining normal systemic iron stores