Radiation and immunotherapy: a synergistic combination

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Immunotherapy can be an effective treatment for metastatic cancer, but a significant subpopulation will not respond, likely due to the lack of antigenic mutations or the immune-evasive properties of cancer. Likewise, radiation therapy (RT) is an established cancer treatment, but local failures still occur. Clinical observations suggest that RT may expand the therapeutic reach of immunotherapy. We examine the immunobiologic and clinical rationale for combining RT and immunotherapy, two modalities yet to be used in combination in routine practice. Preclinical data indicate that RT can potentiate the systemic efficacy of immunotherapy, while activation of the innate and adaptive immune system can enhance the local efficacy of RT.

Authors

Anusha Kalbasi, Carl H. June, Naomi Haas, Neha Vapiwala

AMPK, insulin resistance, and the metabolic syndrome

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Insulin resistance (IR) and hyperinsulinemia are hallmarks of the metabolic syndrome, as are central adiposity, dyslipidemia, and a predisposition to type 2 diabetes, atherosclerotic cardiovascular disease, hypertension, and certain cancers. Regular exercise and calorie restriction have long been known to increase insulin sensitivity and decrease the prevalence of these disorders. The subsequent identification of AMP-activated protein kinase (AMPK) and its activation by exercise and fuel deprivation have led to studies of the effects of AMPK on both IR and metabolic syndrome–related diseases. In this review, we evaluate this body of literature, with special emphasis on the hypothesis that dysregulation of AMPK is both a pathogenic factor for these disorders in humans and a target for their prevention and therapy.

Authors

Neil B. Ruderman, David Carling, Marc Prentki, José M. Cacicedo

A conversation with Christine Seidman

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Abstract

Authors

Ushma S. Neill

Antiinflammatory effects of apoptotic cells

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Apoptotic cells are rapidly phagocytosed by macrophages, a process that represents a critical step in tissue remodeling, immune responses, and the resolution of inflammation. In 1998, Peter Henson, Donna Bratton, and colleagues at National Jewish Health demonstrated that phagocytosis of apoptotic cells actively suppresses inflammation by inhibiting the production of inflammatory cytokines and inducing production of antiinflammatory factors, including TGF-β and prostaglandin E2. Here they discuss the evolving relationship among apoptosis, phagocytosis, and inflammation.

Authors

Peter M. Henson, Donna L. Bratton

Preeclampsia: a link between trophoblast dysregulation and an antiangiogenic state

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A 24-year-old nulliparous woman developed mildly elevated blood pressure (140–150/90–100 mmHg) without proteinuria (20 mg protein in a 24-hour urine collection) at 30^6/7 weeks of gestation. The fetus was small for gestational age (estimated fetal weight under the fifth percentile). At 32^5/7 weeks of gestation, the patient complained of epigastric pain, blood pressure was 180/110 mmHg, proteinuria was documented (780 mg protein in a 24-hour urine collection), schistocytes were detected in the peripheral smear, platelet count was 60,000 cells per mm³, and serum glutamic oxaloacetic transaminase was 234 U/l. The patient was diagnosed with severe preeclampsia/HELLP syndrome. Antenatal steroids were administered to induce fetal lung maturity. She and her family want to know the causes of this condition, what treatment is available, and whether there are any long-term implications of this diagnosis.

Authors

Roberto Romero, Tinnakorn Chaiworapongsa

STALing B cell responses with CD22

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Suppressing unwanted humoral immune responses without compromising the host’s ability to respond to foreign pathogens is a primary goal for therapies aimed at ameliorating harmful autoantibody production. Global suppression of the immune system via lymphocyte depletion and/or immunosuppressive drugs can have off-target effects, a limitation to conventional therapies. In this issue of the JCI, Macauley and colleagues utilize a novel platform to inhibit antigen-specific antibody production that preserves the immune system’s ability to respond to unrelated antigens.

Authors

Craig P. Chappell, Edward A. Clark

Linking MLL and the HGF-MET signaling pathway in liver cancer

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Abstract

Mixed-lineage leukemia (MLL; also known as myeloid/lymphoid), the human homolog of trithorax in Drosophila, is a transcriptional coactivator that plays an essential role during early development and hematopoiesis. Furthermore, MLL is critically involved in the epigenetic regulation of cell cycle, senescence, DNA damage, and stem cell self-renewal. Chromosomal aberrations of MLL in acute leukemias are well documented, but the role of this gene in solid malignancies remains unclear. In this issue of the JCI, Takeda et al. describe a novel epigenetic link between MLL and the HGF-MET signaling pathway conferring invasive and metastatic properties to hepatocellular carcinoma cells.

Authors

Jens U. Marquardt, Snorri S. Thorgeirsson

Mutation signature of adenoid cystic carcinoma: evidence for transcriptional and epigenetic reprogramming

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Adenoid cystic carcinoma (ACC), a relatively rare malignancy usually of salivary gland origin, has a signature v-myb avian myeloblastosis viral oncogene homolog–nuclear factor I/B (MYB-NFIB) gene fusion that activates MYB transcriptional regulatory activity. A new study in this issue by Stephens et al. is a comprehensive genomic mutation profiling analysis of this neoplasm and documents a common theme of alteration in chromatin regulatory genes. Also, mutations in SPEN (split ends, homolog of Drosophila), which encodes an RNA-binding coregulatory protein, suggest that other changes in transcriptional regulation may involve the NOTCH, FGFR, or other signaling pathways in which SPEN participates. Since there is a low prevalence of mutations in common oncogenes and tumor-suppressor genes, it is likely that alterations primarily in specific transcriptional regulatory genes, augmented by changes in chromatin structure, drive the neoplastic process in ACC.

Authors

Henry F. Frierson Jr., Christopher A. Moskaluk

Prenatal sensitization of a postnatal trigger for metabolic disease

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Intrahepatic cholestasis of pregnancy (ICP), marked by elevated maternal serum bile acid levels, occurs in late pregnancy and is often associated with poor perinatal outcomes. In this issue of the JCI, Papacleovoulou et al. analyze the long-term consequences of ICP and find that teens born to mothers with ICP exhibit enhanced characteristics of metabolic syndrome relative to controls. The authors also used a new ICP mouse model to support and extend these findings, demonstrating that in utero exposure to bile acids induces persistent epigenetic alterations and abnormal placental lipogenesis, setting the stage for later metabolic dysfunction.

Authors

Susan K. Murphy

Mitochondrial TCA cycle intermediates regulate body fluid and acid-base balance

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Intrarenal control mechanisms play an important role in the maintenance of body fluid and electrolyte balance and pH homeostasis. Recent discoveries of new ion transport and regulatory pathways in the distal nephron and collecting duct system have helped to better our understanding of these critical kidney functions and identified new potential therapeutic targets and approaches. In this issue of the JCI, Tokonami et al. report on the function of an exciting new paracrine mediator, the mitochondrial the citric acid (TCA) cycle intermediate α-ketoglutarate (αKG), which via its OXGR1 receptor plays an unexpected, nontraditional role in the adaptive regulation of renal HCO₃^– secretion and salt reabsorption.

Authors

János Peti-Peterdi

FcγRIIb mediates amyloid-β neurotoxicity and memory impairment in Alzheimer’s disease

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Amyloid-β (Aβ) induces neuronal loss and cognitive deficits and is believed to be a prominent cause of Alzheimer’s disease (AD); however, the cellular pathology of the disease is not fully understood. Here, we report that IgG Fcγ receptor II-b (FcγRIIb) mediates Aβ neurotoxicity and neurodegeneration. We found that FcγRIIb is significantly upregulated in the hippocampus of AD brains and neuronal cells exposed to synthetic Aβ. Neuronal FcγRIIb activated ER stress and caspase-12, and Fcgr2b KO primary neurons were resistant to synthetic Aβ-induced cell death in vitro. Fcgr2b deficiency ameliorated Aβ-induced inhibition of long-term potentiation and inhibited the reduction of synaptic density by naturally secreted Aβ. Moreover, genetic depletion of Fcgr2b rescued memory impairments in an AD mouse model. To determine the mechanism of action of FcγRIIb in Aβ neurotoxicity, we demonstrated that soluble Aβ oligomers interact with FcγRIIb in vitro and in AD brains, and that inhibition of their interaction blocks synthetic Aβ neurotoxicity. We conclude that FcγRIIb has an aberrant, but essential, role in Aβ-mediated neuronal dysfunction.

Authors

Tae-In Kam, Sungmin Song, Youngdae Gwon, Hyejin Park, Ji-Jing Yan, Isak Im, Ji-Woo Choi, Tae-Yong Choi, Jeongyeon Kim, Dong-Keun Song, Toshiyuki Takai, Yong-Chul Kim, Key-Sun Kim, Se-Young Choi, Sukwoo Choi, William L. Klein, Junying Yuan, Yong-Keun Jung

Immune cells control skin lymphatic electrolyte homeostasis and blood pressure

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The skin interstitium sequesters excess Na⁺ and Cl^– in salt-sensitive hypertension. Mononuclear phagocyte system (MPS) cells are recruited to the skin, sense the hypertonic electrolyte accumulation in skin, and activate the tonicity-responsive enhancer-binding protein (TONEBP, also known as NFAT5) to initiate expression and secretion of VEGFC, which enhances electrolyte clearance via cutaneous lymph vessels and increases eNOS expression in blood vessels. It is unclear whether this local MPS response to osmotic stress is important to systemic blood pressure control. Herein, we show that deletion of TonEBP in mouse MPS cells prevents the VEGFC response to a high-salt diet (HSD) and increases blood pressure. Additionally, an antibody that blocks the lymph-endothelial VEGFC receptor, VEGFR3, selectively inhibited MPS-driven increases in cutaneous lymphatic capillary density, led to skin Cl^– accumulation, and induced salt-sensitive hypertension. Mice overexpressing soluble VEGFR3 in epidermal keratinocytes exhibited hypoplastic cutaneous lymph capillaries and increased Na⁺, Cl^–, and water retention in skin and salt-sensitive hypertension. Further, we found that HSD elevated skin osmolality above plasma levels. These results suggest that the skin contains a hypertonic interstitial fluid compartment in which MPS cells exert homeostatic and blood pressure–regulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3–mediated modification of cutaneous lymphatic capillary function.

Authors

Helge Wiig, Agnes Schröder, Wolfgang Neuhofer, Jonathan Jantsch, Christoph Kopp, Tine V. Karlsen, Michael Boschmann, Jennifer Goss, Maija Bry, Natalia Rakova, Anke Dahlmann, Sven Brenner, Olav Tenstad, Harri Nurmi, Eero Mervaala, Hubertus Wagner, Franz-Xaver Beck, Dominik N. Müller, Dontscho Kerjaschki, Friedrich C. Luft, David G. Harrison, Kari Alitalo, Jens Titze

Microglial activation underlies cerebellar deficits produced by repeated cannabis exposure

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Chronic cannabis exposure can lead to cerebellar dysfunction in humans, but the neurobiological mechanisms involved remain incompletely understood. Here, we found that in mice, subchronic administration of the psychoactive component of cannabis, delta9-tetrahydrocannabinol (THC), activated cerebellar microglia and increased the expression of neuroinflammatory markers, including IL-1β. This neuroinflammatory phenotype correlated with deficits in cerebellar conditioned learning and fine motor coordination. The neuroinflammatory phenotype was readily detectable in the cerebellum of mice with global loss of the CB1 cannabinoid receptor (CB1R, Cb1^–/– mice) and in mice lacking CB1R in the cerebellar parallel fibers, suggesting that CB1R downregulation in the cerebellar molecular layer plays a key role in THC-induced cerebellar deficits. Expression of CB2 cannabinoid receptor (CB2R) and Il1b mRNA was increased under neuroinflammatory conditions in activated CD11b-positive microglial cells. Furthermore, administration of the immunosuppressant minocycline or an inhibitor of IL-1β receptor signaling prevented the deficits in cerebellar function in Cb1^–/– and THC-withdrawn mice. Our results suggest that cerebellar microglial activation plays a crucial role in the cerebellar deficits induced by repeated cannabis exposure.

Authors

Laura Cutando, Arnau Busquets-Garcia, Emma Puighermanal, Maria Gomis-González, José María Delgado-García, Agnès Gruart, Rafael Maldonado, Andrés Ozaita

Reciprocal regulation by TLR4 and TGF-β in tumor-initiating stem-like cells

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Abstract

Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133⁺ TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG–dependent TICs were defective in the TGF-β tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-β signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-β signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-β signaling (Spnb2^+/– mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC.

Authors

Chia-Lin Chen, Hidekazu Tsukamoto, Jian-Chang Liu, Claudine Kashiwabara, Douglas Feldman, Linda Sher, Steven Dooley, Samuel W. French, Lopa Mishra, Lydia Petrovic, Joseph H. Jeong, Keigo Machida

Antigen-activated dendritic cells ameliorate influenza A infections

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Influenza A viruses cause significant morbidity and mortality worldwide. There is a need for alternative or adjunct therapies, as resistance to currently used antiviral drugs is emerging rapidly. We tested ligand epitope antigen presentation system (LEAPS) technology as a new immune-based treatment for influenza virus infection in a mouse model. Influenza-J-LEAPS peptides were synthesized by conjugating the binding ligand derived from the β2-microglobulin chain of the human MHC class I molecule (J-LEAPS) with 15 to 30 amino acid–long peptides derived from influenza virus NP, M, or HA proteins. DCs were stimulated with influenza-J-LEAPS peptides (influenza-J-LEAPS) and injected intravenously into infected mice. Antigen-specific LEAPS-stimulated DCs were effective in reducing influenza virus replication in the lungs and enhancing survival of infected animals. Additionally, they augmented influenza-specific T cell responses in the lungs and reduced the severity of disease by limiting excessive cytokine responses, which are known to contribute to morbidity and mortality following influenza virus infection. Our data demonstrate that influenza-J-LEAPS–pulsed DCs reduce virus replication in the lungs, enhance survival, and modulate the protective immune responses that eliminate the virus while preventing excessive cytokines that could injure the host. This approach shows promise as an adjunct to antiviral treatment of influenza virus infections.

Authors

Kobporn Boonnak, Leatrice Vogel, Marlene Orandle, Daniel Zimmerman, Eyal Talor, Kanta Subbarao

Reversal of gene dysregulation in cultured cytotrophoblasts reveals possible causes of preeclampsia

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During human pregnancy, a subset of placental cytotrophoblasts (CTBs) differentiates into cells that aggressively invade the uterus and its vasculature, anchoring the progeny and rerouting maternal blood to the placenta. In preeclampsia (PE), CTB invasion is limited, reducing placental perfusion and/or creating intermittent flow. This syndrome, affecting 4%–8% of pregnancies, entails maternal vascular alterations (e.g., high blood pressure, proteinuria, and edema) and, in some patients, fetal growth restriction. The only cure is removal of the faulty placenta, i.e., delivery. Previously, we showed that defective CTB differentiation contributes to the placental component of PE, but the causes were unknown. Here, we cultured CTBs isolated from PE and control placentas for 48 hours, enabling differentiation and invasion. In various severe forms of PE, transcriptomics revealed common aberrations in CTB gene expression immediately after isolation, including upregulation of SEMA3B, which resolved in culture. The addition of SEMA3B to normal CTBs inhibited invasion and recreated aspects of the PE phenotype. Additionally, SEMA3B downregulated VEGF signaling through the PI3K/AKT and GSK3 pathways, effects that were observed in PE CTBs. We propose that, in severe PE, the in vivo environment dysregulates CTB gene expression; the autocrine actions of the upregulated molecules (including SEMA3B) impair CTB differentiation, invasion and signaling; and patient-specific factors determine the signs.

Authors

Yan Zhou, Matthew J. Gormley, Nathan M. Hunkapiller, Mirhan Kapidzic, Yana Stolyarov, Victoria Feng, Masakazu Nishida, Penelope M. Drake, Katherine Bianco, Fei Wang, Michael T. McMaster, Susan J. Fisher

CD4⁺ follicular helper T cell infiltration predicts breast cancer survival

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CD4⁺ T cells are critical regulators of immune responses, but their functional role in human breast cancer is relatively unknown. The goal of this study was to produce an image of CD4⁺ T cells infiltrating breast tumors using limited ex vivo manipulation to better understand the in vivo differences associated with patient prognosis. We performed comprehensive molecular profiling of infiltrating CD4⁺ T cells isolated from untreated invasive primary tumors and found that the infiltrating T cell subpopulations included follicular helper T (Tfh) cells, which have not previously been found in solid tumors, as well as Th1, Th2, and Th17 effector memory cells and Tregs. T cell signaling pathway alterations included a mixture of activation and suppression characterized by restricted cytokine/chemokine production, which inversely paralleled lymphoid infiltration levels and could be reproduced in activated donor CD4⁺ T cells treated with primary tumor supernatant. A comparison of extensively versus minimally infiltrated tumors showed that CXCL13-producing CD4⁺ Tfh cells distinguish extensive immune infiltrates, principally located in tertiary lymphoid structure germinal centers. An 8-gene Tfh signature, signifying organized antitumor immunity, robustly predicted survival or preoperative response to chemotherapy. Our identification of CD4⁺ Tfh cells in breast cancer suggests that they are an important immune element whose presence in the tumor is a prognostic factor.

Authors

Chunyan Gu-Trantien, Sherene Loi, Soizic Garaud, Carole Equeter, Myriam Libin, Alexandre de Wind, Marie Ravoet, Hélène Le Buanec, Catherine Sibille, Germain Manfouo-Foutsop, Isabelle Veys, Benjamin Haibe-Kains, Sandeep K. Singhal, Stefan Michiels, Françoise Rothé, Roberto Salgado, Hugues Duvillier, Michail Ignatiadis, Christine Desmedt, Dominique Bron, Denis Larsimont, Martine Piccart, Christos Sotiriou, Karen Willard-Gallo

Transcription factor ATF3 links host adaptive response to breast cancer metastasis

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Host response to cancer signals has emerged as a key factor in cancer development; however, the underlying molecular mechanism is not well understood. In this report, we demonstrate that activating transcription factor 3 (ATF3), a hub of the cellular adaptive response network, plays an important role in host cells to enhance breast cancer metastasis. Immunohistochemical analysis of patient tumor samples revealed that expression of ATF3 in stromal mononuclear cells, but not cancer epithelial cells, is correlated with worse clinical outcomes and is an independent predictor for breast cancer death. This finding was corroborated by data from mouse models showing less efficient breast cancer metastasis in Atf3-deficient mice than in WT mice. Further, mice with myeloid cell–selective KO of Atf3 showed fewer lung metastases, indicating that host ATF3 facilitates metastasis, at least in part, by its function in macrophage/myeloid cells. Gene profiling analyses of macrophages from mouse tumors identified an ATF3-regulated gene signature that could distinguish human tumor stroma from distant stroma and could predict clinical outcomes, lending credence to our mouse models. In conclusion, we identified ATF3 as a regulator in myeloid cells that enhances breast cancer metastasis and has predictive value for clinical outcomes.

Authors

Chris C. Wolford, Stephen J. McConoughey, Swati P. Jalgaonkar, Marino Leon, Anand S. Merchant, Johnna L. Dominick, Xin Yin, Yiseok Chang, Erik J. Zmuda, Sandra A. O’Toole, Ewan K.A. Millar, Stephanie L. Roller, Charles L. Shapiro, Michael C. Ostrowski, Robert L. Sutherland, Tsonwin Hai

Metabolic stress regulates cytoskeletal dynamics and metastasis of cancer cells

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Metabolic reprogramming is an important driver of tumor progression; however, the metabolic regulators of tumor cell motility and metastasis are not understood. Here, we show that tumors maintain energy production under nutrient deprivation through the function of HSP90 chaperones compartmentalized in mitochondria. Using cancer cell lines, we found that mitochondrial HSP90 proteins, including tumor necrosis factor receptor–associated protein-1 (TRAP-1), dampen the activation of the nutrient-sensing AMPK and its substrate UNC-51–like kinase (ULK1), preserve cytoskeletal dynamics, and release the cell motility effector focal adhesion kinase (FAK) from inhibition by the autophagy initiator FIP200. In turn, this results in enhanced tumor cell invasion in low nutrients and metastatic dissemination to bone or liver in disease models in mice. Moreover, we found that phosphorylated ULK1 levels were correlated with shortened overall survival in patients with non–small cell lung cancer. These results demonstrate that mitochondrial HSP90 chaperones, including TRAP-1, overcome metabolic stress and promote tumor cell metastasis by limiting the activation of the nutrient sensor AMPK and preventing autophagy.

Authors

M. Cecilia Caino, Young Chan Chae, Valentina Vaira, Stefano Ferrero, Mario Nosotti, Nina M. Martin, Ashani Weeraratna, Michael O’Connell, Danielle Jernigan, Alessandro Fatatis, Lucia R. Languino, Silvano Bosari, Dario C. Altieri

Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis

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The mechanisms by which deregulated nuclear factor erythroid-2–related factor 2 (NRF2) and kelch-like ECH-associated protein 1 (KEAP1) signaling promote cellular proliferation and tumorigenesis are poorly understood. Using an integrated genomics and ¹³C-based targeted tracer fate association (TTFA) study, we found that NRF2 regulates miR-1 and miR-206 to direct carbon flux toward the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle, reprogramming glucose metabolism. Sustained activation of NRF2 signaling in cancer cells attenuated miR-1 and miR-206 expression, leading to enhanced expression of PPP genes. Conversely, overexpression of miR-1 and miR-206 decreased the expression of metabolic genes and dramatically impaired NADPH production, ribose synthesis, and in vivo tumor growth in mice. Loss of NRF2 decreased the expression of the redox-sensitive histone deacetylase, HDAC4, resulting in increased expression of miR-1 and miR-206, and not only inhibiting PPP expression and activity but functioning as a regulatory feedback loop that repressed HDAC4 expression. In primary tumor samples, the expression of miR-1 and miR-206 was inversely correlated with PPP gene expression, and increased expression of NRF2-dependent genes was associated with poor prognosis. Our results demonstrate that microRNA-dependent (miRNA-dependent) regulation of the PPP via NRF2 and HDAC4 represents a novel link between miRNA regulation, glucose metabolism, and ROS homeostasis in cancer cells.

Authors

Anju Singh, Christine Happel, Soumen K. Manna, George Acquaah-Mensah, Julian Carrerero, Sarvesh Kumar, Poonam Nasipuri, Kristopher W. Krausz, Nobunao Wakabayashi, Ruby Dewi, Laszlo G. Boros, Frank J. Gonzalez, Edward Gabrielson, Kwok K. Wong, Geoffrey Girnun, Shyam Biswal

Dependence receptor UNC5D mediates nerve growth factor depletion–induced neuroblastoma regression

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Spontaneous regression of neuroblastoma (NB) resembles the developmentally regulated programmed cell death (PCD) of sympathetic neurons. Regressing tumor cells express high levels of the nerve growth factor (NGF) receptors TRKA and p75^NTR and are dependent on NGF for survival; however, the underlying molecular mechanism remains elusive. Here, we show that UNC5D, a dependence receptor that is directly targeted by p53 family members, is highly expressed in favorable NBs. NGF withdrawal strongly upregulated UNC5D, E2F1, and p53 in human primary favorable NBs. The induced UNC5D was cleaved by caspases 2/3, and the released intracellular fragment translocated into the nucleus and interacted with E2F1 to selectively transactivate the proapoptotic target gene. The cleavage of UNC5D and its induction of apoptosis were strongly inhibited by addition of netrin-1. Unc5d^–/– mice consistently exhibited a significant increase in dorsal root ganglia neurons and resistance to NGF depletion–induced apoptosis in sympathetic neurons compared with wild-type cells. Our data suggest that UNC5D forms a positive feedback loop with p53 and E2F1 to promote NGF dependence–mediated PCD during NB regression.

Authors

Yuyan Zhu, Yuanyuan Li, Seiki Haraguchi, Meng Yu, Miki Ohira, Toshinori Ozaki, Atsuko Nakagawa, Toshikazu Ushijima, Eriko Isogai, Haruhiko Koseki, Yohko Nakamura, Cuize Kong, Patrick Mehlen, Hirofumi Arakawa, Akira Nakagawara

An androgen receptor N-terminal domain antagonist for treating prostate cancer

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Hormone therapies for advanced prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD), but these ultimately fail and the disease progresses to lethal castration-resistant prostate cancer (CRPC). The mechanisms that drive CRPC are incompletely understood, but may involve constitutively active AR splice variants that lack the LBD. The AR N-terminal domain (NTD) is essential for AR activity, but targeting this domain with small-molecule inhibitors is complicated by its intrinsic disorder. Here we investigated EPI-001, a small-molecule antagonist of AR NTD that inhibits protein-protein interactions necessary for AR transcriptional activity. We found that EPI analogs covalently bound the NTD to block transcriptional activity of AR and its splice variants and reduced the growth of CRPC xenografts. These findings suggest that the development of small-molecule inhibitors that bind covalently to intrinsically disordered proteins is a promising strategy for development of specific and effective anticancer agents.

Authors

Jae-Kyung Myung, Carmen A. Banuelos, Javier Garcia Fernandez, Nasrin R. Mawji, Jun Wang, Amy H. Tien, Yu Chi Yang, Iran Tavakoli, Simon Haile, Kate Watt, Iain J. McEwan, Stephen Plymate, Raymond J. Andersen, Marianne D. Sadar

DISC1 and SLC12A2 interaction affects human hippocampal function and connectivity

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Hippocampal development is coordinated by both extracellular factors like GABA neurotransmission and intracellular components like DISC1. We previously reported that SLC12A2-dependent GABA depolarization and DISC1 coregulate hippocampal neuronal development, and 2 SNPs in these genes linked to mRNA expression interactively increase schizophrenia risk. Using functional MRI, we now confirm this biological interaction in vivo by showing in 2 independent samples of healthy individuals (total N = 349) that subjects homozygous for both risk alleles evince dramatically decreased hippocampal area activation (Cohen’s d = 0.78) and connectivity (d = 0.57) during a recognition memory task. These data highlight the importance of epistatic models in understanding genetic association with complex brain phenotypes.

Authors

Joseph H. Callicott, Emer L. Feighery, Venkata S. Mattay, Michael G. White, Qiang Chen, David A.A. Baranger, Karen F. Berman, Bai Lu, Hongjun Song, Guo-li Ming, Daniel R. Weinberger

Whole exome sequencing of adenoid cystic carcinoma

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Adenoid cystic carcinoma (ACC) is a rare malignancy that can occur in multiple organ sites and is primarily found in the salivary gland. While the identification of recurrent fusions of the MYB-NFIB genes have begun to shed light on the molecular underpinnings, little else is known about the molecular genetics of this frequently fatal cancer. We have undertaken exome sequencing in a series of 24 ACC to further delineate the genetics of the disease. We identified multiple mutated genes that, combined, implicate chromatin deregulation in half of cases. Further, mutations were identified in known cancer genes, including PIK3CA, ATM, CDKN2A, SF3B1, SUFU, TSC1, and CYLD. Mutations in NOTCH1/2 were identified in 3 cases, and we identify the negative NOTCH signaling regulator, SPEN, as a new cancer gene in ACC with mutations in 5 cases. Finally, the identification of 3 likely activating mutations in the tyrosine kinase receptor FGFR2, analogous to those reported in ovarian and endometrial carcinoma, point to potential therapeutic avenues for a subset of cases.

Authors

Philip J. Stephens, Helen R. Davies, Yoshitsugu Mitani, Peter Van Loo, Adam Shlien, Patrick S. Tarpey, Elli Papaemmanuil, Angela Cheverton, Graham R. Bignell, Adam P. Butler, John Gamble, Stephen Gamble, Claire Hardy, Jonathan Hinton, Mingming Jia, Alagu Jayakumar, David Jones, Calli Latimer, Stuart McLaren, David J. McBride, Andrew Menzies, Laura Mudie, Mark Maddison, Keiran Raine, Serena Nik-Zainal, Sarah O’Meara, Jon W. Teague, Ignacio Varela, David C. Wedge, Ian Whitmore, Scott M. Lippman, Ultan McDermott, Michael R. Stratton, Peter J. Campbell, Adel K. El-Naggar, P. Andrew Futreal

PRKDC mutations in a SCID patient with profound neurological abnormalities

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The DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by PRKDC) functions in DNA non-homologous end-joining (NHEJ), the major DNA double strand break (DSB) rejoining pathway. NHEJ also functions during lymphocyte development, joining V(D)J recombination intermediates during antigen receptor gene assembly. Here, we describe a patient with compound heterozygous mutations in PRKDC, low DNA-PKcs expression, barely detectable DNA-PK kinase activity, and impaired DSB repair. In a heterologous expression system, we found that one of the PRKDC mutations inactivated DNA-PKcs, while the other resulted in dramatically diminished but detectable residual function. The patient suffered SCID with reduced or absent T and B cells, as predicted from PRKDC-deficient animal models. Unexpectedly, the patient was also dysmorphic; showed severe growth failure, microcephaly, and seizures; and had profound, globally impaired neurological function. MRI scans revealed microcephaly-associated cortical and hippocampal dysplasia and progressive atrophy over 2 years of life. These neurological features were markedly more severe than those observed in patients with deficiencies in other NHEJ proteins. Although loss of DNA-PKcs in mice, dogs, and horses was previously shown not to impair neuronal development, our findings demonstrate a stringent requirement for DNA-PKcs during human neuronal development and suggest that high DNA-PK protein expression is required to sustain efficient pre- and postnatal neurogenesis.

Authors

Lisa Woodbine, Jessica A. Neal, Nanda-Kumar Sasi, Mayuko Shimada, Karen Deem, Helen Coleman, William B. Dobyns, Tomoo Ogi, Katheryn Meek, E. Graham Davies, Penny A. Jeggo

Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus

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Abstract

Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here, we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP), a molecule externalized in the NETs. NZM mice were treated with Cl-amidine, an inhibitor of peptidylarginine deiminases (PAD), to block NET formation and were evaluated for lupus-like disease activity, endothelial function, and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further, Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells, improved endothelium-dependent vasorelaxation, and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall, these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients.

Authors

Jason S. Knight, Wenpu Zhao, Wei Luo, Venkataraman Subramanian, Alexander A. O’Dell, Srilakshmi Yalavarthi, Jeffrey B. Hodgin, Daniel T. Eitzman, Paul R. Thompson, Mariana J. Kaplan

CpG-depleted adeno-associated virus vectors evade immune detection

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Abstract

Due to their efficient transduction potential, adeno-associated virus (AAV) vectors are leading candidates for gene therapy in skeletal muscle diseases. However, immune responses toward the vector or transgene product have been observed in preclinical and clinical studies. TLR9 has been implicated in promoting AAV-directed immune responses, but vectors have not been developed to circumvent this barrier. To assess the requirement of TLR9 in promoting immunity toward AAV-associated antigens following skeletal muscle gene transfer in mice, we compared immunological responses in WT and Tlr9-deficient mice that received an AAV vector with an immunogenic capsid, AAVrh32.33. In Tlr9-deficient mice, IFN-γ T cell responses toward capsid and transgene antigen were suppressed, resulting in minimal cellular infiltrate and stable transgene expression in target muscles. These findings suggest that AAV-directed immune responses may be circumvented by depleting the ligand for TLR9 (CpG sequences) from the vector genome. Indeed, we found that CpG-depleted AAVrh32.33 vectors could establish persistent transgene expression, evade immunity, and minimize infiltration of effector cells. Thus, CpG-depleted AAV vectors could improve outcome of clinical trials of gene therapy for skeletal muscle disease.

Authors

Susan M. Faust, Peter Bell, Benjamin J. Cutler, Scott N. Ashley, Yanqing Zhu, Joseph E. Rabinowitz, James M. Wilson

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

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Abstract

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Na_vs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na_vs remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Na_vs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury–induced neuropathic pain was used, and an Na_v1.7-specific inhibitor, ProTxII, allowed the isolation of Na_v1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na_v1.7 and Na_v1.8 currents. The redistribution of Na_v1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L^–/–). SNS-Nedd4L^–/– mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na_v1.7 and Na_v1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na_vs and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Authors

Cédric J. Laedermann, Matthieu Cachemaille, Guylène Kirschmann, Marie Pertin, Romain-Daniel Gosselin, Isabelle Chang, Maxime Albesa, Chris Towne, Bernard L. Schneider, Stephan Kellenberger, Hugues Abriel, Isabelle Decosterd

WAVE1 mediates suppression of phagocytosis by phospholipid-derived DAMPs

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Abstract

Clearance of invading pathogens is essential to preventing overwhelming inflammation and sepsis that are symptomatic of bacterial peritonitis. Macrophages participate in this innate immune response by engulfing and digesting pathogens, a process called phagocytosis. Oxidized phospholipids (OxPL) are danger-associated molecular patterns (DAMPs) generated in response to infection that can prevent the phagocytic clearance of bacteria. We investigated the mechanism underlying OxPL action in macrophages. Exposure to OxPL induced alterations in actin polymerization, resulting in spreading of peritoneal macrophages and diminished uptake of E. coli. Pharmacological and cell-based studies showed that an anchored pool of PKA mediates the effects of OxPL. Gene silencing approaches identified the A-kinase anchoring protein (AKAP) WAVE1 as an effector of OxPL action in vitro. Chimeric Wave1^–/– mice survived significantly longer after infection with E. coli and OxPL treatment in vivo. Moreover, we found that endogenously generated OxPL in human peritoneal dialysis fluid from end-stage renal failure patients inhibited phagocytosis via WAVE1. Collectively, these data uncover an unanticipated role for WAVE1 as a critical modulator of the innate immune response to severe bacterial infections.

Authors

Ulrich Matt, Omar Sharif, Rui Martins, Tanja Furtner, Lorene Langeberg, Riem Gawish, Immanuel Elbau, Ana Zivkovic, Karin Lakovits, Olga Oskolkova, Bianca Doninger, Andreas Vychytil, Thomas Perkmann, Gernot Schabbauer, Christoph J. Binder, Valery N. Bochkov, John D. Scott, Sylvia Knapp

Type 2 alveolar cells are stem cells in adult lung

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Abstract

Gas exchange in the lung occurs within alveoli, air-filled sacs composed of type 2 and type 1 epithelial cells (AEC2s and AEC1s), capillaries, and various resident mesenchymal cells. Here, we use a combination of in vivo clonal lineage analysis, different injury/repair systems, and in vitro culture of purified cell populations to obtain new information about the contribution of AEC2s to alveolar maintenance and repair. Genetic lineage-tracing experiments showed that surfactant protein C–positive (SFTPC-positive) AEC2s self renew and differentiate over about a year, consistent with the population containing long-term alveolar stem cells. Moreover, if many AEC2s were specifically ablated, high-resolution imaging of intact lungs showed that individual survivors undergo rapid clonal expansion and daughter cell dispersal. Individual lineage-labeled AEC2s placed into 3D culture gave rise to self-renewing “alveolospheres,” which contained both AEC2s and cells expressing multiple AEC1 markers, including HOPX, a new marker for AEC1s. Growth and differentiation of the alveolospheres occurred most readily when cocultured with primary PDGFRα⁺ lung stromal cells. This population included lipofibroblasts that normally reside close to AEC2s and may therefore contribute to a stem cell niche in the murine lung. Results suggest that a similar dynamic exists between AEC2s and mesenchymal cells in the human lung.

Authors

Christina E. Barkauskas, Michael J. Cronce, Craig R. Rackley, Emily J. Bowie, Douglas R. Keene, Barry R. Stripp, Scott H. Randell, Paul W. Noble, Brigid L.M. Hogan

Loss-of-function mutations in SIM1 contribute to obesity and Prader-Willi–like features

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Abstract

Sim1 haploinsufficiency in mice induces hyperphagic obesity and developmental abnormalities of the brain. In humans, abnormalities in chromosome 6q16, a region that includes SIM1, were reported in obese children with a Prader-Willi–like syndrome; however, SIM1 involvement in obesity has never been conclusively demonstrated. Here, SIM1 was sequenced in 44 children with Prader-Willi–like syndrome features, 198 children with severe early-onset obesity, 568 morbidly obese adults, and 383 controls. We identified 4 rare variants (p.I128T, p.Q152E, p.R581G, and p.T714A) in 4 children with Prader-Willi–like syndrome features (including severe obesity) and 4 other rare variants (p.T46R, p.E62K, p.H323Y, and p.D740H) in 7 morbidly obese adults. By assessing the carriers’ relatives, we found a significant contribution of SIM1 rare variants to intra-family risk for obesity. We then assessed functional effects of the 8 substitutions on SIM1 transcriptional activities in stable cell lines using luciferase gene reporter assays. Three mutations showed strong loss-of-function effects (p.T46R, p.H323Y, and p.T714A) and were associated with high intra-family risk for obesity, while the variants with mild or no effects on SIM1 activity were not associated with obesity within families. Our genetic and functional studies demonstrate a firm link between SIM1 loss of function and severe obesity associated with, or independent of, Prader-Willi–like features.

Authors

Amélie Bonnefond, Anne Raimondo, Fanny Stutzmann, Maya Ghoussaini, Shwetha Ramachandrappa, David C. Bersten, Emmanuelle Durand, Vincent Vatin, Beverley Balkau, Olivier Lantieri, Violeta Raverdy, François Pattou, Wim Van Hul, Luc Van Gaal, Daniel J. Peet, Jacques Weill, Jennifer L. Miller, Fritz Horber, Anthony P. Goldstone, Daniel J. Driscoll, John B. Bruning, David Meyre, Murray L. Whitelaw, Philippe Froguel

Rare variants in single-minded 1 (SIM1) are associated with severe obesity

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Abstract

Single-minded 1 (SIM1) is a basic helix-loop-helix transcription factor involved in the development and function of the paraventricular nucleus of the hypothalamus. Obesity has been reported in Sim1 haploinsufficient mice and in a patient with a balanced translocation disrupting SIM1. We sequenced the coding region of SIM1 in 2,100 patients with severe, early onset obesity and in 1,680 controls. Thirteen different heterozygous variants in SIM1 were identified in 28 unrelated severely obese patients. Nine of the 13 variants significantly reduced the ability of SIM1 to activate a SIM1-responsive reporter gene when studied in stably transfected cells coexpressing the heterodimeric partners of SIM1 (ARNT or ARNT2). SIM1 variants with reduced activity cosegregated with obesity in extended family studies with variable penetrance. We studied the phenotype of patients carrying variants that exhibited reduced activity in vitro. Variant carriers exhibited increased ad libitum food intake at a test meal, normal basal metabolic rate, and evidence of autonomic dysfunction. Eleven of the 13 probands had evidence of a neurobehavioral phenotype. The phenotypic similarities between patients with SIM1 deficiency and melanocortin 4 receptor (MC4R) deficiency suggest that some of the effects of SIM1 deficiency on energy homeostasis are mediated by altered melanocortin signaling.

Authors

Shwetha Ramachandrappa, Anne Raimondo, Anna M.G. Cali, Julia M. Keogh, Elana Henning, Sadia Saeed, Amanda Thompson, Sumedha Garg, Elena G. Bochukova, Soren Brage, Victoria Trowse, Eleanor Wheeler, Adrienne E. Sullivan, Mehul Dattani, Peter E. Clayton, Vippan Datta, John B. Bruning, Nick J. Wareham, Stephen O’Rahilly, Daniel J. Peet, Ines Barroso, Murray L. Whitelaw, I. Sadaf Farooqi

11β-Hydroxysteroid dehydrogenase blockade prevents age-induced skin structure and function defects

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Abstract

Glucocorticoid (GC) excess adversely affects skin integrity, inducing thinning and impaired wound healing. Aged skin, particularly that which has been photo-exposed, shares a similar phenotype. Previously, we demonstrated age-induced expression of the GC-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in cultured human dermal fibroblasts (HDFs). Here, we determined 11β-HSD1 levels in human skin biopsies from young and older volunteers and examined the aged 11β-HSD1 KO mouse skin phenotype. 11β-HSD1 activity was elevated in aged human and mouse skin and in PE compared with donor-matched photo-protected human biopsies. Age-induced dermal atrophy with deranged collagen structural organization was prevented in 11β-HSD1 KO mice, which also exhibited increased collagen density. We found that treatment of HDFs with physiological concentrations of cortisol inhibited rate-limiting steps in collagen biosynthesis and processing. Furthermore, topical 11β-HSD1 inhibitor treatment accelerated healing of full-thickness mouse dorsal wounds, with improved healing also observed in aged 11β-HSD1 KO mice. These findings suggest that elevated 11β-HSD1 activity in aging skin leads to increased local GC generation, which may account for adverse changes occurring in the elderly, and 11β-HSD1 inhibitors may be useful in the treatment of age-associated impairments in dermal integrity and wound healing.

Authors

Ana Tiganescu, Abd A. Tahrani, Stuart A. Morgan, Marcela Otranto, Alexis Desmoulière, Lianne Abrahams, Zaki Hassan-Smith, Elizabeth A. Walker, Elizabeth H. Rabbitt, Mark S. Cooper, Kurt Amrein, Gareth G. Lavery, Paul M. Stewart

Nanoparticle clearance is governed by Th1/Th2 immunity and strain background

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Abstract

Extended circulation of nanoparticles in blood is essential for most clinical applications. Nanoparticles are rapidly cleared by cells of the mononuclear phagocyte system (MPS). Approaches such as grafting polyethylene glycol onto particles (PEGylation) extend circulation times; however, these particles are still cleared, and the processes involved in this clearance remain poorly understood. Here, we present an intravital microscopy–based assay for the quantification of nanoparticle clearance, allowing us to determine the effect of mouse strain and immune system function on particle clearance. We demonstrate that mouse strains that are prone to Th1 immune responses clear nanoparticles at a slower rate than Th2-prone mice. Using depletion strategies, we show that both granulocytes and macrophages participate in the enhanced clearance observed in Th2-prone mice. Macrophages isolated from Th1 strains took up fewer particles in vitro than macrophages from Th2 strains. Treating macrophages from Th1 strains with cytokines to differentiate them into M2 macrophages increased the amount of particle uptake. Conversely, treating macrophages from Th2 strains with cytokines to differentiate them into M1 macrophages decreased their particle uptake. Moreover, these results were confirmed in human monocyte–derived macrophages, suggesting that global immune regulation has a significant impact on nanoparticle clearance in humans.

Authors

Stephen W. Jones, Reid A. Roberts, Gregory R. Robbins, Jillian L. Perry, Marc P. Kai, Kai Chen, Tao Bo, Mary E. Napier, Jenny P.Y. Ting, Joseph M. DeSimone, James E. Bear

Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis

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Abstract

Antibodies confer humoral immunity but can also be harmful when they target an autoantigen, alloantigen, allergen, or biotherapeutic. New strategies are needed for antigen-specific suppression of undesired antibody responses, particularly to T cell–dependent protein antigens, because they elicit T cell help. Here we show that liposomal nanoparticles, displaying both antigen and glycan ligands of the inhibitory coreceptor CD22, induce a tolerogenic program that selectively causes apoptosis in mouse and human B cells. These SIGLEC-engaging tolerance-inducing antigenic liposomes (STALs, where SIGLEC is defined as sialic acid–binding Ig-like lectin) induced robust antigen-specific tolerance to protein antigens in mice, preventing subsequent immune response to challenge with the same antigen. Since development of inhibitory antibodies to FVIII is a serious problem in treatment of hemophilia A patients, we investigated the potential of this approach for inducing tolerance to FVIII in a hemophilia mouse model. STALs prevented formation of inhibitory FVIII antibodies, allowing for effective administration of FVIII to hemophilia mice to prevent bleeding. These findings suggest that STALs could be used to eliminate or prevent harmful B cell–mediated immune responses.

Authors

Matthew S. Macauley, Fabian Pfrengle, Christoph Rademacher, Corwin M. Nycholat, Andrew J. Gale, Annette von Drygalski, James C. Paulson

Sirtuin 1 inhibition delays cyst formation in autosomal-dominant polycystic kidney disease

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Abstract

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2 and is characterized by the development of multiple bilateral renal cysts that replace normal kidney tissue. Here, we used Pkd1 mutant mouse models to demonstrate that the nicotinamide adenine dinucleotide–dependent (NAD-dependent) protein deacetylase sirtuin 1 (SIRT1) is involved in the pathophysiology of ADPKD. SIRT1 was upregulated through c-MYC in embryonic and postnatal Pkd1-mutant mouse renal epithelial cells and tissues and could be induced by TNF-α, which is present in cyst fluid during cyst development. Double conditional knockouts of Pkd1 and Sirt1 demonstrated delayed renal cyst formation in postnatal mouse kidneys compared with mice with single conditional knockout of Pkd1. Furthermore, treatment with a pan-sirtuin inhibitor (nicotinamide) or a SIRT1-specific inhibitor (EX-527) delayed cyst growth in Pkd1 knockout mouse embryonic kidneys, Pkd1 conditional knockout postnatal kidneys, and Pkd1 hypomorphic kidneys. Increased SIRT1 expression in Pkd1 mutant renal epithelial cells regulated cystic epithelial cell proliferation through deacetylation and phosphorylation of Rb and regulated cystic epithelial cell death through deacetylation of p53. This newly identified role of SIRT1 signaling in cystic renal epithelial cells provides the opportunity to develop unique therapeutic strategies for ADPKD.

Authors

Xia Zhou, Lucy X. Fan, William E. Sweeney Jr., John M. Denu, Ellis D. Avner, Xiaogang Li

Integrated genetic and epigenetic analysis of childhood acute lymphoblastic leukemia

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Abstract

Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy and is characterized by recurring structural genetic alterations. Previous studies of DNA methylation suggest epigenetic alterations may also be important, but an integrated genome-wide analysis of genetic and epigenetic alterations in ALL has not been performed. We analyzed 137 B-lineage and 30 T-lineage childhood ALL cases using microarray analysis of DNA copy number alterations and gene expression, and genome-wide cytosine methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. We found that the different genetic subtypes of ALL are characterized by distinct DNA methylation signatures that exhibit significant correlation with gene expression profiles. We also identified an epigenetic signature common to all cases, with correlation to gene expression in 65% of these genes, suggesting that a core set of epigenetically deregulated genes is central to the initiation or maintenance of lymphoid transformation. Finally, we identified aberrant methylation in multiple genes also targeted by recurring DNA copy number alterations in ALL, suggesting that these genes are inactivated far more frequently than suggested by structural genomic analyses alone. Together, these results demonstrate subtype- and disease-specific alterations in cytosine methylation in ALL that influence transcriptional activity, and are likely to exert a key role in leukemogenesis.

Authors

Maria E. Figueroa, Shann-Ching Chen, Anna K. Andersson, Letha A. Phillips, Yushan Li, Jason Sotzen, Mondira Kundu, James R. Downing, Ari Melnick, Charles G. Mullighan

HDAC3 is essential for DNA replication in hematopoietic progenitor cells

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Abstract

Histone deacetylase 3 (HDAC3) contributes to the regulation of gene expression, chromatin structure, and genomic stability. Because HDAC3 associates with oncoproteins that drive leukemia and lymphoma, we engineered a conditional deletion allele in mice to explore the physiological roles of Hdac3 in hematopoiesis. We used the Vav-Cre transgenic allele to trigger recombination, which yielded a dramatic loss of lymphoid cells, hypocellular bone marrow, and mild anemia. Phenotypic and functional analysis suggested that Hdac3 was required for the formation of the earliest lymphoid progenitor cells in the marrow, but that the marrow contained 3–5 times more multipotent progenitor cells. Hdac3^–/– stem cells were severely compromised in competitive bone marrow transplantation. In vitro, Hdac3^–/– stem and progenitor cells failed to proliferate, and most cells remained undifferentiated. Moreover, one-third of the Hdac3^–/– stem and progenitor cells were in S phase 2 hours after BrdU labeling in vivo, suggesting that these cells were impaired in transit through the S phase. DNA fiber-labeling experiments indicated that Hdac3 was required for efficient DNA replication in hematopoietic stem and progenitor cells. Thus, Hdac3 is required for the passage of hematopoietic stem/progenitor cells through the S phase, for stem cell functions, and for lymphopoiesis.

Authors

Alyssa R. Summers, Melissa A. Fischer, Kristy R. Stengel, Yue Zhao, Jonathan F. Kaiser, Christina E. Wells, Aubrey Hunt, Srividya Bhaskara, Jessica W. Luzwick, Shilpa Sampathi, Xi Chen, Mary Ann Thompson, David Cortez, Scott W. Hiebert

Dominant protein interactions that influence the pathogenesis of conformational diseases

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Abstract

Misfolding of exportable proteins can trigger endocrinopathies. For example, misfolding of insulin can result in autosomal dominant mutant INS gene–induced diabetes of youth, and misfolding of thyroglobulin can result in autosomal recessive congenital hypothyroidism with deficient thyroglobulin. Both proinsulin and thyroglobulin normally form homodimers; the mutant versions of both proteins misfold in the ER, triggering ER stress, and, in both cases, heterozygosity creates potential for cross-dimerization between mutant and WT gene products. Here, we investigated these two ER-retained mutant secretory proteins and the selectivity of their interactions with their respective WT counterparts. In both cases and in animal models of these diseases, we found that conditions favoring an increased stoichiometry of mutant gene product dominantly inhibited export of the WT partner, while increased relative level of the WT gene product helped to rescue secretion of the mutant partner. Surprisingly, the bidirectional consequences of secretory blockade and rescue occur simultaneously in the same cells. Thus, in the context of heterozygosity, expression level and stability of WT subunits may be a critical factor influencing the effect of protein misfolding on clinical phenotype. These results offer new insight into dominant as well as recessive inheritance of conformational diseases and offer opportunities for the development of new therapies.

Authors

Jordan Wright, Xiaofan Wang, Leena Haataja, Aaron P. Kellogg, Jaemin Lee, Ming Liu, Peter Arvan

Melanoma immunotherapy using mature DCs expressing the constitutive proteasome

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Abstract

Background. Many cancers, including melanoma, exclusively express constitutive proteasomes (cPs) and are unable to express immunoproteasomes (iPs). In contrast, mature DCs used for immunotherapy exclusively express iPs. Since proteasomes generate peptides presented by HLA class I molecules, we hypothesized that mature melanoma antigen–loaded DCs engineered to process antigens through cPs would be superior inducers of antimelanoma immunity in vivo.

Methods. Subjects with metastatic melanoma were vaccinated with mature DCs transfected with RNAs encoding melanoma antigens MART1, MAGE-3, gp100, and tyrosinase. These DCs were derived from monocytes that were untransfected (Arm A; n = 4), transfected with control siRNA (Arm B; n = 3), or transfected with siRNAs targeting the 3 inducible iP subunits (Arm C; n = 5).

Results. Vaccination stimulated antigen-specific T cell responses in all subjects, which peaked after 3–4 vaccinations, but remained elevated in Arm C subjects. Also in Arm C, circulating melanoma cell levels (as detected by quantitative PCR) fell, and T cell lytic activity against autologous melanoma was induced. In HLA-A2⁺ subjects, CD8⁺ T cells that bound tetramers loaded with cP-derived melanoma antigenic peptides were found in the peripheral blood only in Arm C subjects. Of 2 subjects with active disease (both in Arm C), one had a partial clinical response, while the other, who exhibited diffuse dermal and soft tissue metastases, had a complete response.

Conclusion. These results suggest that the efficacy of melanoma DC–based immunotherapy is enhanced when tumor antigen–loaded DCs used for vaccination express cPs.

Trial registration. Clinicaltrials.gov NCT00672542.

Funding. Duke Clinical Research Institute/Duke Translational Medicine Institute, Duke Melanoma Consortium, and Duke University Department of Surgery.

Authors

Jens Dannull, N. Rebecca Haley, Gary Archer, Smita Nair, David Boczkowski, Mark Harper, Nicole De Rosa, Nancy Pickett, Paul J. Mosca, James Burchette, Maria A. Selim, Duane A. Mitchell, John Sampson, Douglas S. Tyler, Scott K. Pruitt

iPSC-derived β cells model diabetes due to glucokinase deficiency

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Abstract

Diabetes is a disorder characterized by loss of β cell mass and/or β cell function, leading to deficiency of insulin relative to metabolic need. To determine whether stem cell–derived β cells recapitulate molecular-physiological phenotypes of a diabetic subject, we generated induced pluripotent stem cells (iPSCs) from subjects with maturity-onset diabetes of the young type 2 (MODY2), which is characterized by heterozygous loss of function of the gene encoding glucokinase (GCK). These stem cells differentiated into β cells with efficiency comparable to that of controls and expressed markers of mature β cells, including urocortin-3 and zinc transporter 8, upon transplantation into mice. While insulin secretion in response to arginine or other secretagogues was identical to that in cells from healthy controls, GCK mutant β cells required higher glucose levels to stimulate insulin secretion. Importantly, this glucose-specific phenotype was fully reverted upon gene sequence correction by homologous recombination. Our results demonstrate that iPSC-derived β cells reflect β cell–autonomous phenotypes of MODY2 subjects, providing a platform for mechanistic analysis of specific genotypes on β cell function.

Authors

Haiqing Hua, Linshan Shang, Hector Martinez, Matthew Freeby, Mary Pat Gallagher, Thomas Ludwig, Liyong Deng, Ellen Greenberg, Charles LeDuc, Wendy K. Chung, Robin Goland, Rudolph L. Leibel, Dieter Egli

HGF-MET signals via the MLL-ETS2 complex in hepatocellular carcinoma

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Abstract

HGF signals through its cognate receptor, MET, to orchestrate diverse biological processes, including cell proliferation, cell fate specification, organogenesis, and epithelial-mesenchymal transition. Mixed-lineage leukemia (MLL), an epigenetic regulator, plays critical roles in cell fate, stem cell, and cell cycle decisions. Here, we describe a role for MLL in the HGF-MET signaling pathway. We found a shared phenotype among Mll^–/–, Hgf^–/–, and Met^–/– mice with common cranial nerve XII (CNXII) outgrowth and myoblast migration defects. Phenotypic analysis demonstrated that MLL was required for HGF-induced invasion and metastatic growth of hepatocellular carcinoma cell lines. HGF-MET signaling resulted in the accumulation of ETS2, which interacted with MLL to transactivate MMP1 and MMP3. ChIP assays demonstrated that activation of the HGF-MET pathway resulted in increased occupancy of the MLL-ETS2 complex on MMP1 and MMP3 promoters, where MLL trimethylated histone H3 lysine 4 (H3K4), activating transcription. Our results present an epigenetic link between MLL and the HGF-MET signaling pathway, which may suggest new strategies for therapeutic intervention.

Authors

Shugaku Takeda, Han Liu, Satoru Sasagawa, Yiyu Dong, Paul A. Trainor, Emily H. Cheng, James J. Hsieh

α-Ketoglutarate regulates acid-base balance through an intrarenal paracrine mechanism

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Abstract

Paracrine communication between different parts of the renal tubule is increasingly recognized as an important determinant of renal function. Previous studies have shown that changes in dietary acid-base load can reverse the direction of apical α-ketoglutarate (αKG) transport in the proximal tubule and Henle’s loop from reabsorption (acid load) to secretion (base load). Here we show that the resulting changes in the luminal concentrations of αKG are sensed by the αKG receptor OXGR1 expressed in the type B and non-A–non-B intercalated cells of the connecting tubule (CNT) and the cortical collecting duct (CCD). The addition of 1 mM αKG to the tubular lumen strongly stimulated Cl^–-dependent HCO₃^– secretion and electroneutral transepithelial NaCl reabsorption in microperfused CCDs of wild-type mice but not Oxgr1^–/– mice. Analysis of alkali-loaded mice revealed a significantly reduced ability of Oxgr1^–/– mice to maintain acid-base balance. Collectively, these results demonstrate that OXGR1 is involved in the adaptive regulation of HCO₃^– secretion and NaCl reabsorption in the CNT/CCD under acid-base stress and establish αKG as a paracrine mediator involved in the functional coordination of the proximal and the distal parts of the renal tubule.

Authors

Natsuko Tokonami, Luciana Morla, Gabriel Centeno, David Mordasini, Suresh Krishna Ramakrishnan, Svetlana Nikolaeva, Carsten A. Wagner, Olivier Bonny, Pascal Houillier, Alain Doucet, Dmitri Firsov

Maternal cholestasis during pregnancy programs metabolic disease in offspring

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The intrauterine environment is a major contributor to increased rates of metabolic disease in adults. Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy that affects 0.5%–2% of pregnant women and is characterized by increased bile acid levels in the maternal serum. The influence of ICP on the metabolic health of offspring is unknown. We analyzed the Northern Finland birth cohort 1985–1986 database and found that 16-year-old children of mothers with ICP had altered lipid profiles. Males had increased BMI, and females exhibited increased waist and hip girth compared with the offspring of uncomplicated pregnancies. We further investigated the effect of maternal cholestasis on the metabolism of adult offspring in the mouse. Females from cholestatic mothers developed a severe obese, diabetic phenotype with hepatosteatosis following a Western diet, whereas matched mice not exposed to cholestasis in utero did not. Female littermates were susceptible to metabolic disease before dietary challenge. Human and mouse studies showed an accumulation of lipids in the fetoplacental unit and increased transplacental cholesterol transport in cholestatic pregnancy. We believe this is the first report showing that cholestatic pregnancy in the absence of altered maternal BMI or diabetes can program metabolic disease in the offspring.

Authors

Georgia Papacleovoulou, Shadi Abu-Hayyeh, Evanthia Nikolopoulou, Oscar Briz, Bryn M. Owen, Vanya Nikolova, Caroline Ovadia, Xiao Huang, Marja Vaarasmaki, Marc Baumann, Eugene Jansen, Christiane Albrecht, Marjo-Riitta Jarvelin, Jose J.G. Marin, A.S. Knisely, Catherine Williamson

Host immunity contributes to the anti-melanoma activity of BRAF inhibitors

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Abstract

Authors

Deborah A. Knight, Shin Foong Ngiow, Ming Li, Tiffany Parmenter, Stephen Mok, Ashley Cass, Nicole M. Haynes, Kathryn Kinross, Hideo Yagita, Richard C. Koya, Thomas G. Graeber, Antoni Ribas, Grant A. McArthur, Mark J. Smyth

Steroids and osteoporosis: the quest for mechanisms

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Abstract

Authors

Stavros C. Manolagas

GSK3β regulates physiological migration of stem/progenitor cells via cytoskeletal rearrangement

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Abstract

Authors

Kfir Lapid, Tomer Itkin, Gabriele D’Uva, Yossi Ovadya, Aya Ludin, Giulia Caglio, Alexander Kalinkovich, Karin Golan, Ziv Porat, Massimo Zollo, Tsvee Lapidot

Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer

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Abstract

Authors

Sampurna Chatterjee, Lukas C. Heukamp, Maike Siobal, Jakob Schöttle, Caroline Wieczorek, Martin Peifer, Davide Frasca, Mirjam Koker, Katharina König, Lydia Meder, Daniel Rauh, Reinhard Buettner, Jürgen Wolf, Rolf A. Brekken, Bernd Neumaier, Gerhard Christofori, Roman K. Thomas, Roland T. Ullrich