Browsing by Author "Chan, Rebecca J."

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    Cleaning up the environment in juvenile myelomonocytic leukemia
    (AME Publishing Company., 2017-02) Deng, Lisa; Chan, Rebecca J.; Medical and Molecular Genetics, School of Medicine
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    Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis
    (2011-01) Li, Deqiang; Hallett, Mark A.; Zhu, Wuqiang; Rubart, Michael; Liu, Ying; Yang, Zhenyun; Chen, Hanying; Haneline, Laura S.; Chan, Rebecca J.; Schwartz, Robert J.; Field, Loren J.; Atkinson, Simon J.; Shou, Weinian
    Dishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, plays an important role in regulating the actin cytoskeleton via mediation of linear actin assembly. Previous functional studies of Daam1 in lower species suggest its essential role in Drosophila trachea formation and Xenopus gastrulation. However, its in vivo physiological function in mammalian systems is largely unknown. We have generated Daam1-deficient mice via gene-trap technology and found that Daam1 is highly expressed in developing murine organs, including the heart. Daam1-deficient mice exhibit embryonic and neonatal lethality and suffer multiple cardiac defects, including ventricular noncompaction, double outlet right ventricles and ventricular septal defects. In vivo genetic rescue experiments further confirm that the lethality of Daam1-deficient mice results from the inherent cardiac abnormalities. In-depth analyses have revealed that Daam1 is important for regulating filamentous actin assembly and organization, and consequently for cytoskeletal function in cardiomyocytes, which contributes to proper heart morphogenesis. Daam1 is also found to be important for proper cytoskeletal architecture and functionalities in embryonic fibroblasts. Biochemical analyses indicate that Daam1 does not regulate cytoskeletal organization through RhoA, Rac1 or Cdc42. Our study highlights a crucial role for Daam1 in regulating the actin cytoskeleton and tissue morphogenesis.
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    Effect of Systemic Iron Overload and a Chelation Therapy in a Mouse Model of the Neurodegenerative Disease Hereditary Ferritinopathy
    (Plos, 2016-08-30) Garringer, Holly J.; Irimia, Jose M.; Li, Wei; Goodwin, Charles B.; Richine, Briana; Acton, Anthony; Chan, Rebecca J.; Peacock, Munro; Muhoberac, Barry B.; Ghetti, Bernardino; Vidal, Ruben; Department of Pathology and Laboratory Medicine, IU School of Medicine
    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.
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    Erythropoietin stimulates murine and human fibroblast growth factor-23, revealing novel roles for bone and bone marrow
    (Ferrata Storti Foundation, 2017-11) Clinkenbeard, Erica L.; Hanudel, Mark R.; Stayrook, Keith R.; Appaiah, Hitesh Nidumanda; Farrow, Emily G.; Cass, Taryn A.; Summers, Lelia J.; Ip, Colin S.; Hum, Julia M.; Thomas, Joseph C.; Ivan, Mircea; Richine, Briana M.; Chan, Rebecca J.; Clemens, Thomas L.; Schipani, Ernestina; Sabbagh, Yves; Xu, Linlin; Srour, Edward F.; Alvarez, Marta B.; Kacena, Melissa A.; Salusky, Isidro B.; Ganz, Tomas; Nemeth, Elizabeta; White, Kenneth E.; Medical and Molecular Genetics, School of Medicine
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    GCN5-B is a Novel Nuclear Histone Acetyltransferase that is Crucial for Viability in the Protozoan Parasite Toxoplasma gondii
    (2011-03-16) Dixon, Stacey E.; Sullivan, William J., Jr.; Chan, Rebecca J.; Hocevar, Barbara A.; Queener, Sherry F.; Zhang, Jian-Ting
    Infection with the single-celled parasite Toxoplasma gondii (phylum Apicomplexa) is usually benign in normal healthy individuals, but can cause congenital birth defects, ocular disease, and also life-threatening infection in immunocompromised patients. Acute infection caused by tachyzoites is controlled by a healthy immune response, but the parasite differentiates into a latent cyst form (bradyzoite) leading to permanent infection and chronic disease. Current therapies are effective only against tachyzoites, are highly toxic to the patient, and do not eradicate the encysted bradyzoites, thus highlighting the need for novel therapeutics. Inhibitors of histone deacetylases have been shown to reduce parasite viability in vitro demonstrating that chromatin remodeling enzymes, key mediators in epigenetic regulation, might serve as potential drug targets. Furthermore, epigenetic regulation has been shown to contribute to gene expression and differentiation in Toxoplasma. This dissertation focused on investigating the physiological role of a Toxoplasma GCN5-family histone acetyltransferase (HAT), termed TgGCN5-B. It was hypothesized that TgGCN5-B is an essential HAT that resides within a unique, multi-subunit complex in the parasite nucleus. Studies of TgGCN5-B have revealed that this HAT possesses a unique nuclear localization signal (311RPAENKKRGR320) that is both necessary and sufficient to translocate the protein to the parasite nucleus. Although no other protein motifs have been identified in the N-terminal extension of TgGCN5-B, it is likely that this extension plays a role in protein-protein interactions. All GCN5 homologues function within large multi-subunit complexes, many being conserved among species, but bioinformatic analysis of the Toxoplasma genome revealed a lack of many of these conserved components. Biochemical studies identified several potential TgGCN5-B associating proteins, including several novel apicomplexan transcription factors. Preliminary evidence suggested that TgGCN5-B was essential for tachyzoites; therefore, a dominant-negative approach was utilized to examine the role of TgGCN5-B in the physiology of Toxoplasma. When catalytically inactive TgGCN5-B protein was over-expressed in the parasites, there was a significant decrease in tachyzoite growth and viability, with initial observations suggesting defects in nuclear division and daughter cell budding. These results demonstrate that TgGCN5-B is important for tachyzoite development and indicate that therapeutic targeting of this HAT could be a novel approach to treat toxoplasmosis.
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    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
    (Office of the Vice Chancellor for Research, 2011-04-08) Goodwin, Charles B.; Yang, Zhenyun; Yin, Fuqin; Chan, Rebecca J.
    Juvenile 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.
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    Hematopoietic-restricted Ptpn11E76K reveals indolent MPN progression in mice
    (Impact Journals, 2018-04-24) Tarnawsky, Stefan P.; Yu, Wen-Mei; Qu, Cheng-Kui; Chan, Rebecca J.; Yoder, Mervin C.; Biochemistry and Molecular Biology, School of Medicine
    Juvenile Myelomonocytic Leukemia (JMML) is a pediatric myeloproliferative neoplasm (MPN) that has a poor prognosis. Somatic mutations in Ptpn11 are the most frequent cause of JMML and they commonly occur in utero. Animal models of mutant Ptpn11 have probed the signaling pathways that contribute to JMML. However, existing models may inappropriately exacerbate MPN features by relying on non-hematopoietic-restricted Cre-loxP strains or transplantations into irradiated recipients. In this study we generate hematopoietic-restricted models of Ptpn11E76K-mediated disease using Csf1r-MCM and Flt3Cre. We show that these animals have indolent MPN progression despite robust GM-CSF hypersensitivity and Ras-Erk hyperactivation. Rather, the dominant pathology is pronounced thrombocytopenia with expanded extramedullary hematopoiesis. Furthermore, we demonstrate that the timing of tamoxifen administration in Csf1r-MCM mice can specifically induce recombinase activity in either fetal or adult hematopoietic progenitors. We take advantage of this technique to show more rapid monocytosis following Ptpn11E76K expression in fetal progenitors compared with adult progenitors. Finally, we demonstrate that Ptpn11E76K results in the progressive reduction of T cells, most notably of CD4+ and naïve T cells. This corresponds to an increased frequency of T cell progenitors in the thymus and may help explain the occasional emergence of T-cell leukemias in JMML patients. Overall, our study is the first to describe the consequences of hematopoietic-restricted Ptpn11E76K expression in the absence of irradiation. Our techniques can be readily adapted by other researchers studying somatically-acquired blood disorders.
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    Inhibition of BTK and PI3Kδ impairs the development of human JMML stem and progenitor cells
    (Elsevier, 2022) Ramdas, Baskar; Yuen, Lisa Deng; Palam, Lakshmi Reddy; Patel, Roshini; Pasupuleti, Santhosh Kumar; Jideonwo, Victoria; Zhang, Ji; Maguire, Callista; Wong, Eric; Kanumuri, Rahul; Zhang, Chujing; Sandusky, George; Chan, Rebecca J.; Zhang, Chi; Stieglitz, Elliot; Haneline, Laura; Kapur, Reuben; Pediatrics, School of Medicine
    Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasia that lacks effective targeted chemotherapies. Clinically, JMML manifests as monocytic leukocytosis, splenomegaly with consequential thrombocytopenia. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in PTPN11 (SHP2), leading to Erk and Akt hyperactivation. Mechanism(s) involved in co-regulation of Erk and Akt in the context of GOF SHP2 are poorly understood. Here, we show that Bruton’s tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2-bearing cells and utilizes B cell adaptor for PI3K to cooperate with p110δ, the catalytic subunit of PI3K. Dual inhibition of BTK and p110δ reduces the activation of both Erk and Akt. In vivo, individual targeting of BTK or p110δ in a mouse model of human JMML equally reduces monocytosis and splenomegaly; however, the combined treatment results in a more robust inhibition and uniquely rescues anemia and thrombocytopenia. RNA-seq analysis of drug-treated mice showed a profound reduction in the expression of genes associated with leukemic cell migration and inflammation, leading to correction in the infiltration of leukemic cells in the lung, liver, and spleen. Remarkably, in a patient derived xenograft model of JMML, leukemia-initiating stem and progenitor cells were potently inhibited in response to the dual drug treatment.
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    Inhibition of the Gab2/PI3K/mTOR signaling ameliorates myeloid malignancy caused by Ptpn11 (Shp2) gain-of-function mutations
    (Springer Nature, 2017-06) Liu, Wei; Yu, Wen-Mei; Zhang, Jing; Chan, Rebecca J.; Loh, Mignon L.; Zhang, Zheng; Bunting, Kevin D.; Qu, Cheng-Kui; Pediatrics, School of Medicine
    Activating mutations, such as E76K and D61Y, in PTPN11 (SHP2), a protein tyrosine phosphatase implicated in multiple cell signaling processes, are associated with 35% of patients with juvenile myelomonocytic leukemia (JMML), an aggressive childhood myeloproliferative neoplasm (MPN). Here we show that the interaction between leukemia-associated mutant Shp2 and Gab2, a scaffolding protein important for cytokine-induced PI3K/Akt signaling, was enhanced, and that the mTOR pathway was elevated in Ptpn11E76K/+ leukemic cells. Importantly, MPN induced by the Ptpn11E76K/+ mutation was markedly attenuated in Ptpn11E76K/+/Gab2-/- double mutant mice-overproduction of myeloid cells was alleviated, splenomegaly was diminished and myeloid cell infiltration in nonhematopoietic organs was decreased in these double mutants. Excessive myeloid differentiation of stem cells was also normalized by depletion of Gab2. Acute leukemia progression of MPN was reduced in the double mutant mice and, as such, their survival was much prolonged. Furthermore, treatment of Ptpn11E76K/+ mice with Rapamycin, a specific and potent mTOR inhibitor, mitigated MPN phenotypes. Collectively, this study reveals an important role of the Gab2/PI3K/mTOR pathway in mediating the pathogenic signaling of the PTPN11 gain-of-function mutations and a therapeutic potential of Rapamycin for PTPN11 mutation-associated JMML.
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    Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice
    (2011-11-15) Farrow, Emily G.; Yu, Xijie; Summers, Leila J.; Davis, Siobhan I.; Fleet, James C.; Allen, Matthew R.; Robling, Alexander G.; Stayrook, Keith R.; Jideonwo, Victoria; Magers, Martin J.; Garringer, Holly J.; Vidal, Ruben; Chan, Rebecca J.; Goodwin, Charles B.; Hui, Siu L.; Peacock, Munro; White, Kenneth E.
    Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 (176)RXXR(179)/S(180) proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene-environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.