Browsing by Subject "Ras"

Now showing 1 - 9 of 9
  • Thumbnail Image
    Item
    A Collaborative Model for Accelerating the Discovery and Translation of Cancer Therapies
    (American Association for Cancer Research, 2017-11-01) Maertens, Ophélia; McCurrach, Mila E.; Braun, Benjamin S.; De Raedt, Thomas; Epstein, Inbal; Huang, Tannie Q.; Lauchle, Jennifer O.; Lee, Hyerim; Wu, Jianqiang; Cripe, Timothy P.; Clapp, D. Wade; Ratner, Nancy; Shannon, Kevin; Cichowski, Karen; Pediatrics, School of Medicine
    Preclinical studies using genetically engineered mouse models (GEMM) have the potential to expedite the development of effective new therapies; however, they are not routinely integrated into drug development pipelines. GEMMs may be particularly valuable for investigating treatments for less common cancers, which frequently lack alternative faithful models. Here, we describe a multicenter cooperative group that has successfully leveraged the expertise and resources from philanthropic foundations, academia, and industry to advance therapeutic discovery and translation using GEMMs as a preclinical platform. This effort, known as the Neurofibromatosis Preclinical Consortium (NFPC), was established to accelerate new treatments for tumors associated with neurofibromatosis type 1 (NF1). At its inception, there were no effective treatments for NF1 and few promising approaches on the horizon. Since 2008, participating laboratories have conducted 95 preclinical trials of 38 drugs or combinations through collaborations with 18 pharmaceutical companies. Importantly, these studies have identified 13 therapeutic targets, which have inspired 16 clinical trials. This review outlines the opportunities and challenges of building this type of consortium and highlights how it can accelerate clinical translation. We believe that this strategy of foundation-academic-industry partnering is generally applicable to many diseases and has the potential to markedly improve the success of therapeutic development.
  • Thumbnail Image
    Item
    Epac activation sensitizes rat sensory neurons through activation of Ras
    (Elsevier, 2016-01) Shariati, Behzad; Thompson, Eric L.; Nicol, Grant D.; Vasko, Michael R.; Department of Pharmacology and Toxicology, IU School of Medicine
    Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2'-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization.
  • Thumbnail Image
    Item
    Feasibility of using NF1-GRD and AAV for gene replacement therapy in NF1-associated tumors
    (Springer Nature, 2019-06) Bai, Ren-Yuan; Esposito, Dominic; Tam, Ada J.; McCormick, Frank; Riggins, Gregory J.; Clapp, D. Wade; Staedtke, Verena; Pediatrics, School of Medicine
    Neurofibromatosis type 1, including the highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), is featured by the loss of functional neurofibromin 1 (NF1) protein resulting from genetic alterations. A major function of NF1 is suppressing Ras activities, which is conveyed by an intrinsic GTPase-activating protein-related domain (GRD). In this study, we explored the feasibility of restoring Ras GTPase via exogenous expression of various GRD constructs, via gene delivery using a panel of adeno-associated virus (AAV) vectors in MPNST and human Schwann cells (HSCs). We demonstrated that several AAV serotypes achieved favorable transduction efficacies in those cells and a membrane-targeting GRD fused with an H-Ras C-terminal motif (C10) dramatically inhibited the Ras pathway and MPNST cells in a NF1-specific manner. Our results opened up a venue of gene replacement therapy in NF1-related tumors.
  • Thumbnail Image
    Item
    Frs2α and Shp2 signal independently of Gab to mediate FGF signaling in lens development
    (Company of Biologists, 2014-02-01) Li, Hongge; Tao, Chenqi; Cai, Zhigang; Hertzler-Schaefer, Kristina; Collins, Tamica N.; Wang, Fen; Feng, Gen-Sheng; Gotoh, Noriko; Zhang, Xin; Department of Medical and Molecular Genetics, IU School of Medicine
    Fibroblast growth factor (FGF) signaling requires a plethora of adaptor proteins to elicit downstream responses, but the functional significances of these docking proteins remain controversial. In this study, we used lens development as a model to investigate Frs2α and its structurally related scaffolding proteins, Gab1 and Gab2, in FGF signaling. We show that genetic ablation of Frs2α alone has a modest effect, but additional deletion of tyrosine phosphatase Shp2 causes a complete arrest of lens vesicle development. Biochemical evidence suggests that this Frs2α-Shp2 synergy reflects their epistatic relationship in the FGF signaling cascade, as opposed to compensatory or parallel functions of these two proteins. Genetic interaction experiments further demonstrate that direct binding of Shp2 to Frs2α is necessary for activation of ERK signaling, whereas constitutive activation of either Shp2 or Kras signaling can compensate for the absence of Frs2α in lens development. By contrast, knockout of Gab1 and Gab2 failed to disrupt FGF signaling in vitro and lens development in vivo. These results establish the Frs2α-Shp2 complex as the key mediator of FGF signaling in lens development.
  • Thumbnail Image
    Item
    Neurofibromin Deficiency Induces Endothelial Cell Proliferation and Retinal Neovascularization
    (Association for Research in Vision and Ophthalmology, 2018-05-01) Zhang, Hanfang; Hudson, Farlyn Z.; Xu, Zhimin; Tritz, Rebekah; Rojas, Modesto; Patel, Chintan; Haigh, Stephen B.; Bordán, Zsuzsanna; Ingram, David A.; Fulton, David J.; Weintraub, Neal L.; Caldwell, Ruth B.; Stansfield, Brian K.; Medicine, School of Medicine
    Purpose: Neurofibromatosis type 1 (NF1) is the result of inherited mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. Eye manifestations are common in NF1 with recent reports describing a vascular dysplasia in the retina and choroid. Common features of NF1 retinopathy include tortuous and dilated feeder vessels that terminate in capillary tufts, increased endothelial permeability, and neovascularization. Given the retinal vascular phenotype observed in persons with NF1, we hypothesize that preserving neurofibromin may be a novel strategy to control pathologic retinal neovascularization. Methods: Nf1 expression in human endothelial cells (EC) was reduced using small hairpin (sh) RNA and EC proliferation, migration, and capacity to form vessel-like networks were assessed in response to VEGF and hypoxia. Wild-type (WT), Nf1 heterozygous (Nf1+/-), and Nf1flox/+;Tie2cre pups were subjected to hyperoxia/hypoxia using the oxygen-induced retinopathy model. Retinas were analyzed quantitatively for extent of retinal vessel dropout, neovascularization, and capillary branching. Results: Neurofibromin expression was suppressed in response to VEGF, which corresponded with activation of Mek-Erk and PI3-K-Akt signaling. Neurofibromin-deficient EC exhibited enhanced proliferation and network formation in response to VEGF and hypoxia via an Akt-dependent mechanism. In response to hyperoxia/hypoxia, Nf1+/- retinas exhibited increased vessel dropout and neovascularization when compared with WT retinas. Neovascularization was similar between Nf1+/- and Nf1flox/+;Tie2cre retinas, but capillary drop out in Nf1flox/+;Tie2cre retinas was significantly reduced when compared with Nf1+/- retinas. Conclusions: These data suggest that neurofibromin expression is essential for controlling endothelial cell proliferation and retinal neovascularization and therapies targeting neurofibromin-deficient EC may be beneficial.
  • Thumbnail Image
    Item
    Neurofibromin is a novel regulator of Ras-induced reactive oxygen species production in mice and humans
    (Elsevier, 2016-08) Bessler, Waylan K.; Hudson, Farlyn Z.; Zhang, Hanfang; Harris, Valerie; Wang, Yusi; Mund, Julie A.; Downing, Brandon; Ingram, David A., Jr; Case, Jamie; Fulton, David J.; Stansfield, Brian K.; Pediatrics, School of Medicine
    Neurofibromatosis type 1 (NF1) predisposes individuals to early and debilitating cardiovascular disease. Loss of function mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin, leads to accelerated p21(Ras) activity and phosphorylation of multiple downstream kinases, including Erk and Akt. Nf1 heterozygous (Nf1(+/-)) mice develop a robust neointima that mimics human disease. Monocytes/macrophages play a central role in NF1 arterial stenosis as Nf1 mutations in myeloid cells alone are sufficient to reproduce the enhanced neointima observed in Nf1(+/-) mice. Though the molecular mechanisms underlying NF1 arterial stenosis remain elusive, macrophages are important producers of reactive oxygen species (ROS) and Ras activity directly regulates ROS production. Here, we use compound mutant and lineage-restricted mice to demonstrate that Nf1(+/-) macrophages produce excessive ROS, which enhance Nf1(+/-) smooth muscle cell proliferation in vitro and in vivo. Further, use of a specific NADPH oxidase-2 inhibitor to limit ROS production prevents neointima formation in Nf1(+/-) mice. Finally, mononuclear cells from asymptomatic NF1 patients have increased oxidative DNA damage, an indicator of chronic exposure to oxidative stress. These data provide genetic and pharmacologic evidence that excessive exposure to oxidant species underlie NF1 arterial stenosis and provide a platform for designing novels therapies and interventions.
  • Thumbnail Image
    Item
    NEUROFIBROMIN, NERVE GROWTH FACTOR AND RAS: THEIR ROLES IN CONTROLLING THE EXCITABILITY OF MOUSE SENSORY NEURONS
    (2007-01-03T18:34:09Z) Wang, Yue; Nicol, Grant D.; Vasko, Michael R.; Clapp, D. Wade; Cummins, Theodore R.
    ABSTRACT Yue Wang Neurofibromin, nerve growth factor and Ras: their roles in controlling the excitability of mouse sensory neurons Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates the conversion of active Ras-GTP to inactive Ras-GDP. It is likely that sensory neurons with reduced levels of neurofibromin have augmented Ras-GTP activity. In a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/-), the patch-clamp recording technique is used to investigate the role of neurofibromin in controlling the state of neuronal excitability. Sensory neurons isolated from adult Nf1+/- mice generate more APs in response to a ramp of depolarizing current compared to Nf1+/+ mice. In order to elucidate whether the activation of Ras underlies this augmented excitability, sensory neurons are exposed to nerve growth factor (NGF) that activates Ras. In Nf1+/+ neurons, exposure to NGF increases the production of APs. To examine whether activation of Ras contributes to the NGF-induced sensitization in Nf1+/+ neurons, an antibody that neutralizes Ras activity is internally perfused into neurons. The NGF-mediated augmentation of excitability is suppressed by the Ras-blocking antibody in Nf1+/+ neurons, suggesting the NGF-induced sensitization in Nf1+/+ neurons depends on the activation of Ras. Surprisingly, the excitability of Nf1+/- neurons is not altered by the blocking antibody, suggesting that this enhanced excitability may depend on previous activation of downstream effectors of Ras. To determine the mechanism giving rise to augmented excitability of Nf1+/- neurons, isolated membrane currents are examined. Consistent with the enhanced excitability of Nf1+/- neurons, the peak current density of tetrodotoxin-resistant (TTX-R) and TTX-sensitive (TTX-S) sodium currents (INa) are significantly larger than in Nf1+/+ neurons. Although the voltage for half-maximal activation (V0.5) is not different, there is a significant depolarizing shift in the V0.5 for steady-state inactivation of INa in Nf1+/- neurons. In summary, these results demonstrate that the enhanced production of APs in Nf1+/- neurons results from a larger current amplitude and a depolarized voltage dependence of steady-state inactivation of INa that leads to more sodium channels being available for the subsequent firing of APs. My investigation supports the idea that regulation of channels by the Ras cascade is an important determinant of neuronal excitability. Grant D. Nicol, Ph.D, Chair
  • Thumbnail Image
    Item
    The origin of Juvenile Myelomonocytic Leukemia : Insights from developmental hematopoiesis
    (2017-06) Tarnawsky, Stefan Pasichnyk; Yoder, Mervin C.; Chan, Rebecca J.
    Hematopoiesis proceeds through three developmental phases, each with a unique and indispensable function. The individual roles of these phases in the pathogenesis of blood disorders is unknown. We have adapted murine lineage trace models to identify the relative contributions of embryonic, fetal, and adult hematopoietic phases to the origin of Juvenile Myelomonocytic Leukemia. We hypothesized that the fetal phase would have the most pronounced contribution to the development of JMML, a pediatric myeloproliferative disorder whose disease-initiating somatic mutations occur in utero. Progenitors expressing PTPN11E76K from all three waves were growth hypersensitive to GM-CSF due to hyperactive RAS-ERK signaling. However, fulminant myeloproliferation was only seen in fetal and adult cohorts. We observed equal disease severity in FLT3Cre; PTPN11E76K; ROSA26mTmG and CSF1R-MCM; PTPN11E76K; ROSA26YFP cohorts, which had high and low mutant allele frequencies, respectively. This led to the revelation that all progenitors in the BM niche of mutant animals have equal growth hypersensitivity and RAS-ERK hyperactivation due to non-cell autonomous effects of PTPN11E76K. We further identified that FLT3Cre has hematopoietic-restricted expression, and thereby circumvented morbidity from PTPN11E76K expression in endothelial and stromal cells. This led us to hypothesize that FLT3Cre; KrasG12D; ROSA26mTmG would be the first faithful model of JMML to express this disease-initiating mutation. Indeed, FLT3Cre; KrasG12D mice were born at expected Mendelian ratio and showed normal weight gain to 2 weeks of age. Thereafter, they acquired defining features of JMML including monocytosis, anaemia, thrombocytopenia, and hepatosplenomegaly. All FLT3Cre; KrasG12D mice succumb to a JMML-like disease, which was propagated following transplantation. This is in contrast with CSF1R-MCM; KrasG12D; ROSA26YFP mice, in which low mutant allele frequencies in either fetal or adult HSCs uniformly resulted in T-ALL. Our models reveal previously underappreciated features of JMML including an expansion of dendritic cells and a pronounced defect in T-lymphocyte development. We are the first to demonstrate non-cell autonomous effects of hematopoietic-restricted PTPN11E76K expression. Most importantly, we have shown that both the spatial and the temporal origin of JMML-initiating mutations will affect disease manifestations. Each of our findings suggest novel strategies to treat this intractable disease.
  • Thumbnail Image
    Item
    Small-molecule covalent bond formation at tyrosine creates a binding site and inhibits activation of Ral GTPases
    (National Academy of Sciences, 2020-03-31) Bum-Erdene, Khuchtumur; Liu, Degang; Gonzalez-Gutierrez, Giovanni; Ghozayel, Mona K.; Xu, David; Meroueh, Samy O.; Biochemistry and Molecular Biology, School of Medicine
    Ral (Ras-like) GTPases are directly activated by oncogenic Ras GTPases. Mutant K-Ras (G12C) has enabled the development of covalent K-Ras inhibitors currently in clinical trials. However, Ral, and the overwhelming majority of mutant oncogenic K-Ras, are devoid of a druggable pocket and lack an accessible cysteine for the development of a covalent inhibitor. Here, we report that covalent bond formation by an aryl sulfonyl fluoride electrophile at a tyrosine residue (Tyr-82) inhibits guanine exchange factor Rgl2-mediated nucleotide exchange of Ral GTPase. A high-resolution 1.18-Å X-ray cocrystal structure shows that the compound binds to a well-defined binding site in RalA as a result of a switch II loop conformational change. The structure, along with additional high-resolution crystal structures of several analogs in complex with RalA, confirm the importance of key hydrogen bond anchors between compound sulfone oxygen atoms and Ral backbone nitrogen atoms. Our discovery of a pocket with features found on known druggable sites and covalent modification of a bystander tyrosine residue present in Ral and Ras GTPases provide a strategy that could lead to therapeutic agent targeting oncogenic Ras mutants that are devoid of a cysteine nucleophile.