Browsing by Subject "Mice, Inbred NOD"

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Open Access
A cord blood monocyte-derived cell therapy product accelerates brain remyelination.
(JCI insight, 2016-08-18) Saha, Arjun; Buntz, Susan; Scotland, Paula; Xu, Li; Noeldner, Pamela; Patel, Sachit; Wollish, Amy; Gunaratne, Aruni; Gentry, Tracy; Troy, Jesse; Matsushima, Glenn K; Kurtzberg, Joanne; Balber, Andrew E
Microglia and monocytes play important roles in regulating brain remyelination. We developed DUOC-01, a cell therapy product intended for treatment of demyelinating diseases, from banked human umbilical cord blood (CB) mononuclear cells. Immunodepletion and selection studies demonstrated that DUOC-01 cells are derived from CB CD14⁺ monocytes. We compared the ability of freshly isolated CB CD14⁺ monocytes and DUOC-01 cells to accelerate remyelination of the brains of NOD/SCID/IL2Rγ^null mice following cuprizone feeding-mediated demyelination. The corpus callosum of mice intracranially injected with DUOC-01 showed enhanced myelination, a higher proportion of fully myelinated axons, decreased gliosis and cellular infiltration, and more proliferating oligodendrocyte lineage cells than those of mice receiving excipient. Uncultured CB CD14⁺ monocytes also accelerated remyelination, but to a significantly lesser extent than DUOC-01 cells. Microarray analysis, quantitative PCR studies, Western blotting, and flow cytometry demonstrated that expression of factors that promote remyelination including PDGF-AA, stem cell factor, IGF1, MMP9, MMP12, and triggering receptor expressed on myeloid cells 2 were upregulated in DUOC-01 compared to CB CD14⁺ monocytes. Collectively, our results show that DUOC-01 accelerates brain remyelination by multiple mechanisms and could be beneficial in treating demyelinating conditions.
Open Access
A Transcriptional Signature Identifies LKB1 Functional Status as a Novel Determinant of MEK Sensitivity in Lung Adenocarcinoma.
(Cancer research, 2017-01) Kaufman, Jacob M; Yamada, Tadaaki; Park, Kyungho; Timmers, Cynthia D; Amann, Joseph M; Carbone, David P
LKB1 is a commonly mutated tumor suppressor in non-small cell lung cancer that exerts complex effects on signal transduction and transcriptional regulation. To better understand the downstream impact of loss of functional LKB1, we developed a transcriptional fingerprint assay representing this phenotype. This assay was predictive of LKB1 functional loss in cell lines and clinical specimens, even those without detected sequence alterations in the gene. In silico screening of drug sensitivity data identified putative LKB1-selective drug candidates, revealing novel associations not apparent from analysis of LKB1 mutations alone. Among the candidates, MEK inhibitors showed robust association with signature expression in both training and testing datasets independent of RAS/RAF mutations. This susceptibility phenotype is directly altered by RNA interference-mediated LKB1 knockdown or by LKB1 re-expression into mutant cell lines and is readily observed in vivo using a xenograft model. MEK sensitivity is dependent on LKB1-induced changes in AKT and FOXO3 activation, consistent with genomic and proteomic analyses of LKB1-deficient lung adenocarcinomas. Our findings implicate the MEK pathway as a potential therapeutic target for LKB1-deficient cancers and define a practical NanoString biomarker to identify functional LKB1 loss. Cancer Res; 77(1); 153-63. ©2016 AACR.
Open Access
Copper signaling axis as a target for prostate cancer therapeutics.
(Cancer Res, 2014-10-15) Safi, R; Nelson, ER; Chitneni, SK; Franz, KJ; George, DJ; Zalutsky, MR; McDonnell, DP
Previously published reports indicate that serum copper levels are elevated in patients with prostate cancer and that increased copper uptake can be used as a means to image prostate tumors. It is unclear, however, to what extent copper is required for prostate cancer cell function as we observed only modest effects of chelation strategies on the growth of these cells in vitro. With the goal of exploiting prostate cancer cell proclivity for copper uptake, we developed a "conditional lethal" screen to identify compounds whose cytotoxic actions were manifested in a copper-dependent manner. Emerging from this screen was a series of dithiocarbamates, which, when complexed with copper, induced reactive oxygen species-dependent apoptosis of malignant, but not normal, prostate cells. One of the dithiocarbamates identified, disulfiram (DSF), is an FDA-approved drug that has previously yielded disappointing results in clinical trials in patients with recurrent prostate cancer. Similarly, in our studies, DSF alone had a minimal effect on the growth of prostate cancer tumors when propagated as xenografts. However, when DSF was coadministered with copper, a very dramatic inhibition of tumor growth in models of hormone-sensitive and of castrate-resistant disease was observed. Furthermore, we determined that prostate cancer cells express high levels of CTR1, the primary copper transporter, and additional chaperones that are required to maintain intracellular copper homeostasis. The expression levels of most of these proteins are increased further upon treatment of androgen receptor (AR)-positive prostate cancer cell lines with androgens. Not surprisingly, robust CTR1-dependent uptake of copper into prostate cancer cells was observed, an activity that was accentuated by activation of AR. Given these data linking AR to intracellular copper uptake, we believe that dithiocarbamate/copper complexes are likely to be effective for the treatment of patients with prostate cancer whose disease is resistant to classical androgen ablation therapies.
Open Access
Epigenetic basis of oncogenic-Kras-mediated epithelial-cellular proliferation and plasticity.
(Developmental cell, 2022-02) Kadur Lakshminarasimha Murthy, Preetish; Xi, Rui; Arguijo, Diana; Everitt, Jeffrey I; Kocak, Dewran D; Kobayashi, Yoshihiko; Bozec, Aline; Vicent, Silvestre; Ding, Shengli; Crawford, Gregory E; Hsu, David; Tata, Purushothama Rao; Reddy, Timothy; Shen, Xiling
Oncogenic Kras induces a hyper-proliferative state that permits cells to progress to neoplasms in diverse epithelial tissues. Depending on the cell of origin, this also involves lineage transformation. Although a multitude of downstream factors have been implicated in these processes, the precise chronology of molecular events controlling them remains elusive. Using mouse models, primary human tissues, and cell lines, we show that, in Kras-mutant alveolar type II cells (AEC2), FOSL1-based AP-1 factor guides the mSWI/SNF complex to increase chromatin accessibility at genomic loci controlling the expression of genes necessary for neoplastic transformation. We identified two orthogonal processes in Kras-mutant distal airway club cells. The first promoted their transdifferentiation into an AEC2-like state through NKX2.1, and the second controlled oncogenic transformation through the AP-1 complex. Our results suggest that neoplasms retain an epigenetic memory of their cell of origin through cell-type-specific transcription factors. Our analysis showed that a cross-tissue-conserved AP-1-dependent chromatin remodeling program regulates carcinogenesis.
Open Access
Gene expression-based classifiers identify Staphylococcus aureus infection in mice and humans.
(PLoS One, 2013) Ahn, Sun Hee; Tsalik, Ephraim L; Cyr, Derek D; Zhang, Yurong; van Velkinburgh, Jennifer C; Langley, Raymond J; Glickman, Seth W; Cairns, Charles B; Zaas, Aimee K; Rivers, Emanuel P; Otero, Ronny M; Veldman, Tim; Kingsmore, Stephen F; Kingsmore, Stephen F; Lucas, Joseph; Woods, Christopher W; Ginsburg, Geoffrey S; Fowler, Vance G
Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and uncertainty lead to treatment delays and inappropriate antibiotic use. A growing literature suggests the host's inflammatory response to the pathogen represents a potential tool to improve upon current diagnostics. The hypothesis of this study is that the host responds differently to S. aureus than to E. coli infection in a quantifiable way, providing a new diagnostic avenue. This study uses Bayesian sparse factor modeling and penalized binary regression to define peripheral blood gene-expression classifiers of murine and human S. aureus infection. The murine-derived classifier distinguished S. aureus infection from healthy controls and Escherichia coli-infected mice across a range of conditions (mouse and bacterial strain, time post infection) and was validated in outbred mice (AUC>0.97). A S. aureus classifier derived from a cohort of 94 human subjects distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC 0.99) and E. coli BSI (AUC 0.84). Murine and human responses to S. aureus infection share common biological pathways, allowing the murine model to classify S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers were validated in an independent human cohort (AUC 0.95 and 0.92, respectively). The approach described here lends insight into the conserved and disparate pathways utilized by mice and humans in response to these infections. Furthermore, this study advances our understanding of S. aureus infection; the host response to it; and identifies new diagnostic and therapeutic avenues.
Open Access
Microglia are effector cells of CD47-SIRPα antiphagocytic axis disruption against glioblastoma.
(Proceedings of the National Academy of Sciences of the United States of America, 2019-01) Hutter, Gregor; Theruvath, Johanna; Graef, Claus Moritz; Zhang, Michael; Schoen, Matthew Kenneth; Manz, Eva Maria; Bennett, Mariko L; Olson, Andrew; Azad, Tej D; Sinha, Rahul; Chan, Carmel; Assad Kahn, Suzana; Gholamin, Sharareh; Wilson, Christy; Grant, Gerald; He, Joy; Weissman, Irving L; Mitra, Siddhartha S; Cheshier, Samuel H
Glioblastoma multiforme (GBM) is a highly aggressive malignant brain tumor with fatal outcome. Tumor-associated macrophages and microglia (TAMs) have been found to be major tumor-promoting immune cells in the tumor microenvironment. Hence, modulation and reeducation of tumor-associated macrophages and microglia in GBM is considered a promising antitumor strategy. Resident microglia and invading macrophages have been shown to have distinct origin and function. Whereas yolk sac-derived microglia reside in the brain, blood-derived monocytes invade the central nervous system only under pathological conditions like tumor formation. We recently showed that disruption of the SIRPα-CD47 signaling axis is efficacious against various brain tumors including GBM primarily by inducing tumor phagocytosis. However, most effects are attributed to macrophages recruited from the periphery but the role of the brain resident microglia is unknown. Here, we sought to utilize a model to distinguish resident microglia and peripheral macrophages within the GBM-TAM pool, using orthotopically xenografted, immunodeficient, and syngeneic mouse models with genetically color-coded macrophages (Ccr2 ^RFP) and microglia (Cx3cr1 ^GFP). We show that even in the absence of phagocytizing macrophages (Ccr2 ^RFP/RFP), microglia are effector cells of tumor cell phagocytosis in response to anti-CD47 blockade. Additionally, macrophages and microglia show distinct morphological and transcriptional changes. Importantly, the transcriptional profile of microglia shows less of an inflammatory response which makes them a promising target for clinical applications.
Open Access
Natural allelic variation of the IL-21 receptor modulates ischemic stroke infarct volume.
(The Journal of clinical investigation, 2016-08) Lee, Han Kyu; Keum, Sehoon; Sheng, Huaxin; Warner, David S; Lo, Donald C; Marchuk, Douglas A
Risk for ischemic stroke has a strong genetic basis, but heritable factors also contribute to the extent of damage after a stroke has occurred. We previously identified a locus on distal mouse chromosome 7 that contributes over 50% of the variation in postischemic cerebral infarct volume observed between inbred strains. Here, we used ancestral haplotype analysis to fine-map this locus to 12 candidate genes. The gene encoding the IL-21 receptor (Il21r) showed a marked difference in strain-specific transcription levels and coding variants in neonatal and adult cortical tissue. Collateral vessel connections were moderately reduced in Il21r-deficient mice, and cerebral infarct volume increased 2.3-fold, suggesting that Il21r modulates both collateral vessel anatomy and innate neuroprotection. In brain slice explants, oxygen deprivation (OD) activated apoptotic pathways and increased neuronal cell death in IL-21 receptor-deficient (IL-21R-deficient) mice compared with control animals. We determined that the neuroprotective effects of IL-21R arose from signaling through JAK/STAT pathways and upregulation of caspase 3. Thus, natural genetic variation in murine Il21r influences neuronal cell viability after ischemia by modulating receptor function and downstream signal transduction. The identification of neuroprotective genes based on naturally occurring allelic variations has the potential to inform the development of drug targets for ischemic stroke treatment.
Open Access
Protein tyrosine phosphatase-σ regulates hematopoietic stem cell-repopulating capacity.
(J Clin Invest, 2015-01) Quarmyne, Mamle; Doan, Phuong L; Himburg, Heather A; Yan, Xiao; Nakamura, Mai; Zhao, Liman; Chao, Nelson J; Chute, John P
Hematopoietic stem cell (HSC) function is regulated by activation of receptor tyrosine kinases (RTKs). Receptor protein tyrosine phosphatases (PTPs) counterbalance RTK signaling; however, the functions of receptor PTPs in HSCs remain incompletely understood. We found that a receptor PTP, PTPσ, was substantially overexpressed in mouse and human HSCs compared with more mature hematopoietic cells. Competitive transplantation of bone marrow cells from PTPσ-deficient mice revealed that the loss of PTPσ substantially increased long-term HSC-repopulating capacity compared with BM cells from control mice. While HSCs from PTPσ-deficient mice had no apparent alterations in cell-cycle status, apoptosis, or homing capacity, these HSCs exhibited increased levels of activated RAC1, a RhoGTPase that regulates HSC engraftment capacity. shRNA-mediated silencing of PTPσ also increased activated RAC1 levels in wild-type HSCs. Functionally, PTPσ-deficient BM cells displayed increased cobblestone area-forming cell (CAFC) capacity and augmented transendothelial migration capacity, which was abrogated by RAC inhibition. Specific selection of human cord blood CD34⁺CD38⁻CD45RA⁻lin⁻ PTPσ⁻ cells substantially increased the repopulating capacity of human HSCs compared with CD34⁺CD38⁻CD45RA⁻lin⁻ cells and CD34⁺CD38⁻CD45RA⁻lin⁻PTPσ⁺ cells. Our results demonstrate that PTPσ regulates HSC functional capacity via RAC1 inhibition and suggest that selecting for PTPσ-negative human HSCs may be an effective strategy for enriching human HSCs for transplantation.
Open Access
Survivin as a therapeutic target in Sonic hedgehog-driven medulloblastoma.
(Oncogene, 2015-07) Brun, SN; Markant, SL; Esparza, LA; Garcia, G; Terry, D; Huang, J-M; Pavlyukov, MS; Li, X-N; Grant, GA; Crawford, JR; Levy, ML; Conway, EM; Smith, LH; Nakano, I; Berezov, A; Greene, MI; Wang, Q; Wechsler-Reya, RJ
Medulloblastoma (MB) is a highly malignant brain tumor that occurs primarily in children. Although surgery, radiation and high-dose chemotherapy have led to increased survival, many MB patients still die from their disease, and patients who survive suffer severe long-term side effects as a consequence of treatment. Thus, more effective and less toxic therapies for MB are critically important. Development of such therapies depends in part on identification of genes that are necessary for growth and survival of tumor cells. Survivin is an inhibitor of apoptosis protein that regulates cell cycle progression and resistance to apoptosis, is frequently expressed in human MB and when expressed at high levels predicts poor clinical outcome. Therefore, we hypothesized that Survivin may have a critical role in growth and survival of MB cells and that targeting it may enhance MB therapy. Here we show that Survivin is overexpressed in tumors from patched (Ptch) mutant mice, a model of Sonic hedgehog (SHH)-driven MB. Genetic deletion of survivin in Ptch mutant tumor cells significantly inhibits proliferation and causes cell cycle arrest. Treatment with small-molecule antagonists of Survivin impairs proliferation and survival of both murine and human MB cells. Finally, Survivin antagonists impede growth of MB cells in vivo. These studies highlight the importance of Survivin in SHH-driven MB, and suggest that it may represent a novel therapeutic target in patients with this disease.