Position Statement, Association of American Physicians
The imperative to invest in science has never been greater

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John M. Carethers, Shaun Coughlin, Betty Diamond, Serpil Erzurum, Linda P. Fried, J. Larry Jameson, Kenneth Kaushansky, Mary E. Klotman, Stanley Lemon, Beverly Mitchell, Paul Rothman, Charles Sawyers, Christine Seidman, Stefan Somlo

Conversations with Lasker giants

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Authors

Ushma S. Neill

CXCR3⁺CCR5⁺ T cells and autoimmune diseases: guilty as charged?

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Prior to the 1990s, genetic analyses indicated that many autoimmune diseases are driven by T cell responses; however, the identity of the pathogenic T cell populations responsible for dysfunctional autoimmune responses remained unclear. Some 20 years ago, the discovery of numerous chemokines and their receptors along with the development of specific mAbs to these provided a distinct advance. These new tools revealed a remarkable dichotomy and disclosed that some chemokine receptors guided the constitutive migration of T cells through lymphoid tissues, whereas others, such as CCR5 and CXCR3, guided effector and memory T cell migration to inflammatory lesions. These T cell markers enabled a new understanding of immune responses and the types of T cells involved in different inflammatory reactions.

Authors

Charles R. Mackay

IL-1β inhibition in autoimmune inner ear disease: can you hear me now?

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Clinical vignette: A 51-year-old man with right-sided sudden hearing loss presents to the otology clinic. He has a 4-year history of episodic vertigo of several hours’ duration and fluctuating, progressive sensorineural hearing loss in his left ear. The vertigo attacks have not occurred for the last 18 months, and the left ear hearing is consistently poor. The patient’s right ear hearing has dropped in the last 36 hours. MRI imaging of brain and temporal bone are normal. A 2-week “burst and taper” of oral prednisone is administered with no effect. Over the next 3 months, serial audiograms show rapidly progressive loss of threshold and word recognition scores on the right side. A trial of high-dose prednisone (60 mg/d for 30 days) results in full recovery of the right ear hearing and substantial improvement in the left ear. As the prednisone dose is slowly tapered over several months, the hearing drops again.

Authors

Steven D. Rauch

Too much of a good thing: immunodeficiency due to hyperactive PI3K signaling

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Primary immune deficiency diseases arise due to heritable defects that often involve signaling molecules required for immune cell function. Typically, these genetic defects cause loss of gene function, resulting in primary immune deficiencies such as severe combined immune deficiency (SCID) and X-linked agammaglobulinemia (XLA); however, gain-of-function mutations may also promote immune deficiency. In this issue of the JCI, Deau et al. establish that gain-of-function mutations in PIK3R1, which encodes the p85α regulatory subunit of class IA PI3Ks, lead to immunodeficiency. These observations are consistent with previous reports that hyperactivating mutations in PIK3CD, which encodes the p110δ catalytic subunit, are capable of promoting immune deficiency. Mutations that reduce PI3K activity also result in defective lymphocyte development and function; therefore, these findings support the notion that too little or too much PI3K activity leads to immunodeficiency.

Authors

Craig M. Walsh, David A. Fruman

Play down protein to play up metabolism?

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Who among us hasn’t fantasized about a diet that allows ingestion of a surfeit of calories that are burned off effortlessly by ramping up energy expenditure? In this issue of the JCI, research led by Christopher Morrison suggests that this dream may become a reality; however, a complete understanding of the molecular interface that connects nutrient choices with our cellular metabolism will be required. Laeger et al. show that the expression and secretion of the weight-reducing hormone fibroblast growth factor 21 (FGF21) is regulated by dietary proteins and not, as has been heretofore assumed, simply triggered by reduced caloric intake. This study not only sheds new light on the role of FGF21 in systems metabolism, but also on the ways our bodies cope with the ever-changing availability of different dietary macronutrients.

Authors

Timo D. Müller, Matthias H. Tschöp

Healing the injured vessel wall using microRNA-facilitated gene delivery

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Drug-eluting stents have emerged as potent weapons in the treatment of patients with symptomatic coronary artery disease by reducing restenosis rates; however, a significant clinical consequence of these stents is delayed reendothelialization, which may increase the risk of late stent thrombosis. In this issue of the JCI, Santulli and colleagues generated an adenovirus that expresses the cyclin-dependent kinase inhibitor p27^Kip1 (p27) and bears four tandem copies of target sequences for the endothelial cell–enriched microRNA (miRNA) miR-126-3p (Ad-p27-126TS) in an attempt to specifically reduce proliferation of vascular smooth muscle cells, but not endothelial cells. Indeed, delivery of Ad-p27-126TS to balloon-injured arteries in rats not only induced faster and more complete reendothelialization, but also effectively improved neointimal hyperplasia, hypercoagulability, and vasoreactivity. Collectively, these findings provide a cogent foundation for the potential therapeutic use of miRNA-facilitated gene delivery strategies to heal vessel wall injury.

Authors

Mark W. Feinberg

“RAS”ling β cells to proliferate for diabetes: why do we need MEN?

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Adult human pancreatic β cells are refractory to current therapeutic approaches to enhance proliferation. This reluctance to expand is problematic, especially for people with diabetes who lack sufficient numbers of functional insulin-producing β cells and could therefore benefit from therapies for β cell expansion. In this issue of the JCI, Chamberlain et al. describe a surprising series of observations that involve two downstream arms of the RAS signaling pathway, MAPK and RASSF proteins, which also involve the tumor suppressor menin. The findings of this study may help explain the difficulty of inducing β cell proliferation and may provide leads for therapeutic expansion of human β cells.

Authors

Adolfo García-Ocaña, Andrew F. Stewart

Schlemm’s canal: more than meets the eye, lymphatics in disguise

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Schlemm’s canal (SC) is a unique vascular structure that functions to maintain fluid homeostasis by draining aqueous humor from the eye into the systemic circulation. The endothelium lining the inner wall of SC has both blood and lymphatic vascular characteristics, thus prompting exploration of the development and regulation of this unique channel. In this issue of the JCI, back-to-back papers by Aspelund et al. and Park et al. detail the mechanisms of SC development, which includes a lymphatic reprogramming that is necessary to maintain proper function. Furthermore, both groups exploit the lymph-like qualities of this canal: they identify VEGF-C as a potential therapeutic for glaucoma and suggest that expression of PROX1, a marker of lymphatic fate, could also serve as a biosensor for monitoring SC integrity. These studies provide substantial insight into the molecular and cellular pathways that govern SC development and reveal that ocular pathology is associated with deregulation of the lymph-like characteristics of SC.

Authors

Natalie O. Karpinich, Kathleen M. Caron

Inflammatory lymphangiogenesis in postpartum breast tissue remodeling

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Like many cancers, mammary carcinomas use lymphatic vessels to disseminate, and numerous clinical and experimental studies have documented a strong correlation between peritumoral lymphangiogenesis and tumor dissemination. At the same time, many other factors can affect the incidence, invasiveness, and mortality of breast cancer, including lactation history. Although lactation reduces overall cancer risk, patients diagnosed within 5 years of pregnancy have an increased incidence of metastatic disease. In this issue of the JCI, Lyons and colleagues demonstrate that postpartum breast tissue remodeling during involution coincides with inflammatory lymphangiogenesis. In mouse models, cyclooxygenase-2 (COX-2) inhibition during involution reduced the risk of cancer metastasis and correlated with decreased lymphangiogenesis. In addition to lymphangiogenesis, COX-2 inhibition reduces many of the immune-suppressive features of the tumor microenvironment, including development of myeloid-derived suppressor cells and regulatory T cells; therefore, these results support the notion that inhibiting COX-2 during lactation weaning may lessen the incidence of breast cancer metastasis.

Authors

Melody A. Swartz

Targeted p16^Ink4a epimutation causes tumorigenesis and reduces survival in mice

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Cancer has long been viewed as a genetic disease; however, epigenetic silencing as the result of aberrant promoter DNA methylation is frequently associated with cancer development, suggesting an epigenetic component to the disease. Nonetheless, it has remained unclear whether an epimutation (an aberrant change in epigenetic regulation) can induce tumorigenesis. Here, we exploited a functionally validated cis-acting regulatory element and devised a strategy to induce developmentally regulated genomic targeting of DNA methylation. We used this system to target DNA methylation within the p16^Ink4a promoter in mice in vivo. Engineered p16^Ink4a promoter hypermethylation led to transcriptional suppression in somatic tissues during aging and increased the incidence of spontaneous cancers in these mice. Further, mice carrying a germline p16^Ink4a mutation in one allele and a somatic epimutation in the other had accelerated tumor onset and substantially shortened tumor-free survival. Taken together, these results provide direct functional evidence that p16^Ink4a epimutation drives tumor formation and malignant progression and validate a targeted methylation approach to epigenetic engineering.

Authors

Da-Hai Yu, Robert A. Waterland, Pumin Zhang, Deborah Schady, Miao-Hsueh Chen, Yongtao Guan, Manasi Gadkari, Lanlan Shen

Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes

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The endocannabinoid system (ECS) regulates multiple physiological processes, including cutaneous cell growth and differentiation. Here, we explored the effects of the major nonpsychotropic phytocannabinoid of Cannabis sativa, (-)-cannabidiol (CBD), on human sebaceous gland function and determined that CBD behaves as a highly effective sebostatic agent. Administration of CBD to cultured human sebocytes and human skin organ culture inhibited the lipogenic actions of various compounds, including arachidonic acid and a combination of linoleic acid and testosterone, and suppressed sebocyte proliferation via the activation of transient receptor potential vanilloid-4 (TRPV4) ion channels. Activation of TRPV4 interfered with the prolipogenic ERK1/2 MAPK pathway and resulted in the downregulation of nuclear receptor interacting protein-1 (NRIP1), which influences glucose and lipid metabolism, thereby inhibiting sebocyte lipogenesis. CBD also exerted complex antiinflammatory actions that were coupled to A2a adenosine receptor-dependent upregulation of tribbles homolog 3 (TRIB3) and inhibition of the NF-κB signaling. Collectively, our findings suggest that, due to the combined lipostatic, antiproliferative, and antiinflammatory effects, CBD has potential as a promising therapeutic agent for the treatment of acne vulgaris.

Authors

Attila Oláh, Balázs I. Tóth, István Borbíró, Koji Sugawara, Attila G. Szöllõsi, Gabriella Czifra, Balázs Pál, Lídia Ambrus, Jennifer Kloepper, Emanuela Camera, Matteo Ludovici, Mauro Picardo, Thomas Voets, Christos C. Zouboulis, Ralf Paus, Tamás Bíró

IL-10–producing NKT10 cells are a distinct regulatory invariant NKT cell subset

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Invariant natural killer T (iNKT) cells rapidly produce copious amounts of multiple cytokines after activation, thereby impacting a wide variety of different immune reactions. However, strong activation of iNKT cells with α-galactosylceramide (αGalCer) reportedly induces a hyporeactive state that resembles anergy. In contrast, we determined here that iNKT cells from mice pretreated with αGalCer retain cytotoxic activity and maintain the ability to respond to TCR-dependent as well as TCR-independent cytokine-mediated stimulation. Additionally, αGalCer-pretreated iNKT cells acquired characteristics of regulatory cells, including production and secretion of the immunomodulatory cytokine IL-10. Through the production of IL-10, αGalCer-pretreated iNKT cells impaired antitumor responses and reduced disease in experimental autoimmune encephalomyelitis, a mouse model of autoimmune disease. Furthermore, a subset of iNKT cells with a similar inhibitory phenotype and function were present in mice not exposed to αGalCer and were enriched in mouse adipose tissue and detectable in human PBMCs. These data demonstrate that IL-10–producing iNKT cells with regulatory potential (NKT10 cells) represent a distinct iNKT cell subset.

Authors

Duygu Sag, Petra Krause, Catherine C. Hedrick, Mitchell Kronenberg, Gerhard Wingender

EGFR phosphorylation of DCBLD2 recruits TRAF6 and stimulates AKT-promoted tumorigenesis

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Aberrant activation of EGFR in human cancers promotes tumorigenesis through stimulation of AKT signaling. Here, we determined that the discoidina neuropilin-like membrane protein DCBLD2 is upregulated in clinical specimens of glioblastomas and head and neck cancers (HNCs) and is required for EGFR-stimulated tumorigenesis. In multiple cancer cell lines, EGFR activated phosphorylation of tyrosine 750 (Y750) of DCBLD2, which is located within a recently identified binding motif for TNF receptor-associated factor 6 (TRAF6). Consequently, phosphorylation of DCBLD2 Y750 recruited TRAF6, leading to increased TRAF6 E3 ubiquitin ligase activity and subsequent activation of AKT, thereby enhancing EGFR-driven tumorigenesis. Moreover, evaluation of patient samples of gliomas and HNCs revealed an association among EGFR activation, DCBLD2 phosphorylation, and poor prognoses. Together, our findings uncover a pathway in which DCBLD2 functions as a signal relay for oncogenic EGFR signaling to promote tumorigenesis and suggest DCBLD2 and TRAF6 as potential therapeutic targets for human cancers that are associated with EGFR activation.

Authors

Haizhong Feng, Giselle Y. Lopez, Chung Kwon Kim, Angel Alvarez, Christopher G. Duncan, Ryo Nishikawa, Motoo Nagane, An-Jey A. Su, Philip E. Auron, Matthew L. Hedberg, Lin Wang, Jeffery J. Raizer, John A. Kessler, Andrew T. Parsa, Wei-Qiang Gao, Sung-Hak Kim, Mutsuko Minata, Ichiro Nakano, Jennifer R. Grandis, Roger E. McLendon, Darell D. Bigner, Hui-Kuan Lin, Frank B. Furnari, Webster K. Cavenee, Bo Hu, Hai Yan, Shi-Yuan Cheng

Proteasome function is required for platelet production

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The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1^fl/fl Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1^fl/fl Pdgf-Cre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients.

Authors

Dallas S. Shi, Matthew C.P. Smith, Robert A. Campbell, Patrick W. Zimmerman, Zechariah B. Franks, Bjorn F. Kraemer, Kellie R. Machlus, Jing Ling, Patrick Kamba, Hansjörg Schwertz, Jesse W. Rowley, Rodney R. Miles, Zhi-Jian Liu, Martha Sola-Visner, Joseph E. Italiano Jr., Hilary Christensen, Walter H.A. Kahr, Dean Y. Li, Andrew S. Weyrich

Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett’s metaplasia

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Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barrett’s esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barrett’s metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barrett’s pathogenesis. By microarray analysis, we found that the transcription factor Foxa2 is more highly expressed in murine embryonic esophagus compared with postnatal esophagus. Conditional activation of Shh in mouse esophageal epithelium induced FOXA2, while FOXA2 expression was reduced in Shh knockout embryos, establishing Foxa2 as an esophageal Hh target gene. Evaluation of patient samples revealed FOXA2 expression in Barrett’s metaplasia, dysplasia, and adenocarcinoma but not in esophageal squamous epithelium or squamous cell carcinoma. In esophageal squamous cell lines, Hh signaling upregulated FOXA2, which induced expression of MUC2, an intestinal mucin found in Barrett’s esophagus, and the MUC2-processing protein AGR2. Together, these data indicate that Hh signaling induces expression of genes that determine an intestinal phenotype in esophageal squamous epithelial cells and may contribute to the development of Barrett’s metaplasia.

Authors

David H. Wang, Anjana Tiwari, Monica E. Kim, Nicholas J. Clemons, Nanda L. Regmi, William A. Hodges, David M. Berman, Elizabeth A. Montgomery, D. Neil Watkins, Xi Zhang, Qiuyang Zhang, Chunfa Jie, Stuart J. Spechler, Rhonda F. Souza

Ventromedial hypothalamus–specific Ptpn1 deletion exacerbates diet-induced obesity in female mice

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Protein-tyrosine phosphatase 1B (PTP1B) regulates food intake (FI) and energy expenditure (EE) by inhibiting leptin signaling in the hypothalamus. In peripheral tissues, PTP1B regulates insulin signaling, but its effects on CNS insulin action are largely unknown. Mice harboring a whole-brain deletion of the gene encoding PTP1B (Ptpn1) are lean, leptin-hypersensitive, and resistant to high fat diet–induced (HFD-induced) obesity. Arcuate proopiomelanocortin (POMC) neuron–specific deletion of Ptpn1 causes a similar, but much milder, phenotype, suggesting that PTP1B also acts in other neurons to regulate metabolism. Steroidogenic factor-1–expressing (SF-1–expressing) neurons in the ventromedial hypothalamus (VMH) play an important role in regulating body weight, FI, and EE. Surprisingly, Ptpn1 deletion in SF-1 neurons caused an age-dependent increase in adiposity in HFD-fed female mice. Although leptin sensitivity was increased and FI was reduced in these mice, they had impaired sympathetic output and decreased EE. Immunohistochemical analysis showed enhanced leptin and insulin signaling in VMH neurons from mice lacking PTP1B in SF-1 neurons. Thus, in the VMH, leptin negatively regulates FI, promoting weight loss, whereas insulin suppresses EE, leading to weight gain. Our results establish a novel role for PTP1B in regulating insulin action in the VMH and suggest that increased insulin responsiveness in SF-1 neurons can overcome leptin hypersensitivity and enhance adiposity.

Authors

Franck Chiappini, Karyn J. Catalano, Jennifer Lee, Odile D. Peroni, Jacqueline Lynch, Abha S. Dhaneshwar, Kerry Wellenstein, Alexandra Sontheimer, Benjamin G. Neel, Barbara B. Kahn

Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis

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The intestinal epithelium has a high rate of turnover, and dysregulation of pathways that regulate regeneration can lead to tumor development; however, the negative regulators of oncogenic events in the intestinal epithelium are not fully understood. Here we identified a feedback loop between the epidermal growth factor receptor (EGFR), a known mediator of proliferation, and the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), in intestinal epithelial cells (IECs). We found that TRPV1 was expressed by IECs and was intrinsically activated upon EGFR stimulation. Subsequently, TRPV1 activation inhibited EGFR-induced epithelial cell proliferation via activation of Ca²⁺/calpain and resulting activation of protein tyrosine phosphatase 1B (PTP1B). In a murine model of multiple intestinal neoplasia (Apc^Min/+ mice), TRPV1 deficiency increased adenoma formation, and treatment of these animals with an EGFR kinase inhibitor reversed protumorigenic phenotypes, supporting a functional association between TRPV1 and EGFR signaling in IECs. Administration of a TRPV1 agonist suppressed intestinal tumorigenesis in Apc^Min/+ mice, similar to — as well as in conjunction with — a cyclooxygenase-2 (COX-2) inhibitor, which suggests that targeting both TRPV1 and COX-2 has potential as a therapeutic approach for tumor prevention. Our findings implicate TRPV1 as a regulator of growth factor signaling in the intestinal epithelium through activation of PTP1B and subsequent suppression of intestinal tumorigenesis.

Authors

Petrus R. de Jong, Naoki Takahashi, Alexandra R. Harris, Jihyung Lee, Samuel Bertin, James Jeffries, Michael Jung, Jen Duong, Amy I. Triano, Jongdae Lee, Yaron Niv, David S. Herdman, Koji Taniguchi, Chang-Whan Kim, Hui Dong, Lars Eckmann, Stephanie M. Stanford, Nunzio Bottini, Maripat Corr, Eyal Raz

DEAD-box helicase DP103 defines metastatic potential of human breast cancers

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Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β–activated kinase-1 (TAK1) phosphorylation of NF-κB–activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB–mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.

Authors

Eun Myoung Shin, Hui Sin Hay, Moon Hee Lee, Jen Nee Goh, Tuan Zea Tan, Yin Ping Sen, See Wee Lim, Einas M. Yousef, Hooi Tin Ong, Aye Aye Thike, Xiangjun Kong, Zhengsheng Wu, Earnest Mendoz, Wei Sun, Manuel Salto-Tellez, Chwee Teck Lim, Peter E. Lobie, Yoon Pin Lim, Celestial T. Yap, Qi Zeng, Gautam Sethi, Martin B. Lee, Patrick Tan, Boon Cher Goh, Lance D. Miller, Jean Paul Thiery, Tao Zhu, Louis Gaboury, Puay Hoon Tan, Kam Man Hui, George Wai-Cheong Yip, Shigeki Miyamoto, Alan Prem Kumar, Vinay Tergaonkar

Canonical WNT signaling components in vascular development and barrier formation

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Canonical WNT signaling is required for proper vascularization of the CNS during embryonic development. Here, we used mice with targeted mutations in genes encoding canonical WNT pathway members to evaluate the exact contribution of these components in CNS vascular development and in specification of the blood-brain barrier (BBB) and blood-retina barrier (BRB). We determined that vasculature in various CNS regions is differentially sensitive to perturbations in canonical WNT signaling. The closely related WNT signaling coreceptors LDL receptor–related protein 5 (LRP5) and LRP6 had redundant functions in brain vascular development and barrier maintenance; however, loss of LRP5 alone dramatically altered development of the retinal vasculature. The BBB in the cerebellum and pons/interpeduncular nuclei was highly sensitive to decrements in canonical WNT signaling, and WNT signaling was required to maintain plasticity of barrier properties in mature CNS vasculature. Brain and retinal vascular defects resulting from ablation of Norrin/Frizzled4 signaling were ameliorated by stabilizing β-catenin, while inhibition of β-catenin–dependent transcription recapitulated the vascular development and barrier defects associated with loss of receptor, coreceptor, or ligand, indicating that Norrin/Frizzled4 signaling acts predominantly through β-catenin–dependent transcriptional regulation. Together, these data strongly support a model in which identical or nearly identical canonical WNT signaling mechanisms mediate neural tube and retinal vascularization and maintain the BBB and BRB.

Authors

Yulian Zhou, Yanshu Wang, Max Tischfield, John Williams, Philip M. Smallwood, Amir Rattner, Makoto M. Taketo, Jeremy Nathans

Arachidonate 15-lipoxygenase is required for chronic myeloid leukemia stem cell survival

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Cancer stem cells (CSCs) are responsible for the initiation and maintenance of some types of cancer, suggesting that inhibition of these cells may limit disease progression and relapse. Unfortunately, few CSC-specific genes have been identified. Here, we determined that the gene encoding arachidonate 15-lipoxygenase (Alox15/15-LO) is essential for the survival of leukemia stem cells (LSCs) in a murine model of BCR-ABL–induced chronic myeloid leukemia (CML). In the absence of Alox15, BCR-ABL was unable to induce CML in mice. Furthermore, Alox15 deletion impaired LSC function by affecting cell division and apoptosis, leading to an eventual depletion of LSCs. Moreover, chemical inhibition of 15-LO function impaired LSC function and attenuated CML in mice. The defective CML phenotype in Alox15-deficient animals was rescued by depleting the gene encoding P-selectin, which is upregulated in Alox15-deficient animals. Both deletion and overexpression of P-selectin affected the survival of LSCs. In human CML cell lines and CD34⁺ cells, knockdown of Alox15 or inhibition of 15-LO dramatically reduced survival. Loss of Alox15 altered expression of PTEN, PI3K/AKT, and the transcription factor ICSBP, which are known mediators of cancer pathogenesis. These results suggest that ALOX15 has potential as a therapeutic target for eradicating LSCs in CML.

Authors

Yaoyu Chen, Cong Peng, Sheela A. Abraham, Yi Shan, Zhiru Guo, Ngoc Desouza, Giulia Cheloni, Dongguang Li, Tessa L. Holyoake, Shaoguang Li

VEGF-C–dependent stimulation of lymphatic function ameliorates experimental inflammatory bowel disease

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Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBDs) of unknown etiology that are associated with an aberrant mucosal immune response. Neoangiogenesis and vascular injury are observed in IBD along with increased lymphangiogenesis. While the pathogenic role of angiogenesis in IBD is well characterized, it is not clear how or if increased lymphangiogenesis promotes disease. Here, we determined that enhancing lymphangiogenesis and lymphatic function reduces experimental IBD. Specifically, we demonstrated that adenoviral induction of prolymphangiogenic factor VEGF-C provides marked protection against the development of acute and chronic colitis in 2 different animal models. VEGF-C–dependent protection was observed in combination with increased inflammatory cell mobilization and bacterial antigen clearance from the inflamed colon to the draining lymph nodes. Moreover, we found that the VEGF-C/VEGFR3 pathway regulates macrophage (MΦ) plasticity and activation both in cultured MΦs and in vivo, imparting a hybrid M1-M2 phenotype. The protective function of VEGF-C was meditated by the so-called resolving MΦs during chronic experimental colitis in a STAT6-dependent manner. Together, these findings shed light on the contribution of lymphatics to the pathogenesis of gut inflammation and suggest that correction of defective lymphatic function with VEGF-C has potential as a therapeutic strategy for IBD.

Authors

Silvia D’Alessio, Carmen Correale, Carlotta Tacconi, Alessandro Gandelli, Giovanni Pietrogrande, Stefania Vetrano, Marco Genua, Vincenzo Arena, Antonino Spinelli, Laurent Peyrin-Biroulet, Claudio Fiocchi, Silvio Danese

FCGR2C polymorphisms associate with HIV-1 vaccine protection in RV144 trial

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The phase III RV144 HIV-1 vaccine trial estimated vaccine efficacy (VE) to be 31.2%. This trial demonstrated that the presence of HIV-1–specific IgG-binding Abs to envelope (Env) V1V2 inversely correlated with infection risk, while the presence of Env-specific plasma IgA Abs directly correlated with risk of HIV-1 infection. Moreover, Ab-dependent cellular cytotoxicity responses inversely correlated with risk of infection in vaccine recipients with low IgA; therefore, we hypothesized that vaccine-induced Fc receptor–mediated (FcR-mediated) Ab function is indicative of vaccine protection. We sequenced exons and surrounding areas of FcR-encoding genes and found one FCGR2C tag SNP (rs114945036) that associated with VE against HIV-1 subtype CRF01_AE, with lysine at position 169 (169K) in the V2 loop (CRF01_AE 169K). Individuals carrying CC in this SNP had an estimated VE of 15%, while individuals carrying CT or TT exhibited a VE of 91%. Furthermore, the rs114945036 SNP was highly associated with 3 other FCGR2C SNPs (rs138747765, rs78603008, and rs373013207). Env-specific IgG and IgG3 Abs, IgG avidity, and neutralizing Abs inversely correlated with CRF01_AE 169K HIV-1 infection risk in the CT- or TT-carrying vaccine recipients only. These data suggest a potent role of Fc-γ receptors and Fc-mediated Ab function in conferring protection from transmission risk in the RV144 VE trial.

Authors

Shuying S. Li, Peter B. Gilbert, Georgia D. Tomaras, Gustavo Kijak, Guido Ferrari, Rasmi Thomas, Chul-Woo Pyo, Susan Zolla-Pazner, David Montefiori, Hua-Xin Liao, Gary Nabel, Abraham Pinter, David T. Evans, Raphael Gottardo, James Y. Dai, Holly Janes, Daryl Morris, Youyi Fong, Paul T. Edlefsen, Fusheng Li, Nicole Frahm, Michael D. Alpert, Heather Prentice, Supachai Rerks-Ngarm, Punnee Pitisuttithum, Jaranit Kaewkungwal, Sorachai Nitayaphan, Merlin L. Robb, Robert J. O’Connell, Barton F. Haynes, Nelson L. Michael, Jerome H. Kim, M. Juliana McElrath, Daniel E. Geraghty

Periderm prevents pathological epithelial adhesions during embryogenesis

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Appropriate development of stratified, squamous, keratinizing epithelia, such as the epidermis and oral epithelia, generates an outer protective permeability barrier that prevents water loss, entry of toxins, and microbial invasion. During embryogenesis, the immature ectoderm initially consists of a single layer of undifferentiated, cuboidal epithelial cells that stratifies to produce an outer layer of flattened periderm cells of unknown function. Here, we determined that periderm cells form in a distinct pattern early in embryogenesis, exhibit highly polarized expression of adhesion complexes, and are shed from the outer surface of the embryo late in development. Mice carrying loss-of-function mutations in the genes encoding IFN regulatory factor 6 (IRF6), IκB kinase-α (IKKα), and stratifin (SFN) exhibit abnormal epidermal development, and we determined that mutant animals exhibit dysfunctional periderm formation, resulting in abnormal intracellular adhesions. Furthermore, tissue from a fetus with cocoon syndrome, a lethal disorder that results from a nonsense mutation in IKKA, revealed an absence of periderm. Together, these data indicate that periderm plays a transient but fundamental role during embryogenesis by acting as a protective barrier that prevents pathological adhesion between immature, adhesion-competent epithelia. Furthermore, this study suggests that failure of periderm formation underlies a series of devastating birth defects, including popliteal pterygium syndrome, cocoon syndrome, and Bartsocas-Papas syndrome.

Authors

Rebecca J. Richardson, Nigel L. Hammond, Pierre A. Coulombe, Carola Saloranta, Heidi O. Nousiainen, Riitta Salonen, Andrew Berry, Neil Hanley, Denis Headon, Riitta Karikoski, Michael J. Dixon

Cyclooxygenase-2–dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer

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Breast involution following pregnancy has been implicated in the high rates of metastasis observed in postpartum breast cancers; however, it is not clear how this remodeling process promotes metastasis. Here, we demonstrate that human postpartum breast cancers have increased peritumor lymphatic vessel density that correlates with increased frequency of lymph node metastases. Moreover, lymphatic vessel density was increased in normal postpartum breast tissue compared with tissue from nulliparous women. In rodents, mammary lymphangiogenesis was upregulated during weaning-induced mammary gland involution. Furthermore, breast cancer cells exposed to the involuting mammary microenvironment acquired prolymphangiogenic properties that contributed to peritumor lymphatic expansion, tumor size, invasion, and distant metastases. Finally, in rodent models of postpartum breast cancer, cyclooxygenase-2 (COX-2) inhibition during the involution window decreased normal mammary gland lymphangiogenesis, mammary tumor-associated lymphangiogenesis, tumor cell invasion into lymphatics, and metastasis. Our data indicate that physiologic COX-2–dependent lymphangiogenesis occurs in the postpartum mammary gland and suggest that tumors within this mammary microenvironment acquire enhanced prolymphangiogenic activity. Further, our results suggest that the prolymphangiogenic microenvironment of the postpartum mammary gland has potential as a target to inhibit metastasis and suggest that further study of the therapeutic efficacy of COX-2 inhibitors in postpartum breast cancer is warranted.

Authors

Traci R. Lyons, Virginia F. Borges, Courtney B. Betts, Qiuchen Guo, Puja Kapoor, Holly A. Martinson, Sonali Jindal, Pepper Schedin

FGF21 is an endocrine signal of protein restriction

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Abstract

Enhanced fibroblast growth factor 21 (FGF21) production and circulation has been linked to the metabolic adaptation to starvation. Here, we demonstrated that hepatic FGF21 expression is induced by dietary protein restriction, but not energy restriction. Circulating FGF21 was increased 10-fold in mice and rats fed a low-protein (LP) diet. In these animals, liver Fgf21 expression was increased within 24 hours of reduced protein intake. In humans, circulating FGF21 levels increased dramatically following 28 days on a LP diet. LP-induced increases in FGF21 were associated with increased phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the liver, and both baseline and LP-induced serum FGF21 levels were reduced in mice lacking the eIF2α kinase general control nonderepressible 2 (GCN2). Finally, while protein restriction altered food intake, energy expenditure, and body weight gain in WT mice, FGF21-deficient animals did not exhibit these changes in response to a LP diet. These and other data demonstrate that reduced protein intake underlies the increase in circulating FGF21 in response to starvation and a ketogenic diet and that FGF21 is required for behavioral and metabolic responses to protein restriction. FGF21 therefore represents an endocrine signal of protein restriction, which acts to coordinate metabolism and growth during periods of reduced protein intake.

Authors

Thomas Laeger, Tara M. Henagan, Diana C. Albarado, Leanne M. Redman, George A. Bray, Robert C. Noland, Heike Münzberg, Susan M. Hutson, Thomas W. Gettys, Michael W. Schwartz, Christopher D. Morrison

A human immunodeficiency caused by mutations in the PIK3R1 gene

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Abstract

Recently, patient mutations that activate PI3K signaling have been linked to a primary antibody deficiency. Here, we used whole-exome sequencing and characterized the molecular defects in 4 patients from 3 unrelated families diagnosed with hypogammaglobulinemia and recurrent infections. We identified 2 different heterozygous splice site mutations that affect the same splice site in PIK3R1, which encodes the p85α subunit of PI3K. The resulting deletion of exon 10 produced a shortened p85α protein that lacks part of the PI3K p110-binding domain. The hypothetical loss of p85α-mediated inhibition of p110 activity was supported by elevated phosphorylation of the known downstream signaling kinase AKT in patient T cell blasts. Analysis of patient blood revealed that naive T and memory B cell counts were low, and T cell blasts displayed enhanced activation-induced cell death, which was corrected by addition of the PI3Kδ inhibitor IC87114. Furthermore, B lymphocytes proliferated weakly in response to activation via the B cell receptor and TLR9, indicating a B cell defect. The phenotype exhibited by patients carrying the PIK3R1 splice site mutation is similar to that of patients carrying gain-of-function mutations in PIK3CD. Our results suggest that PI3K activity is tightly regulated in T and B lymphocytes and that various defects in the PI3K-triggered pathway can cause primary immunodeficiencies.

Authors

Marie-Céline Deau, Lucie Heurtier, Pierre Frange, Felipe Suarez, Christine Bole-Feysot, Patrick Nitschke, Marina Cavazzana, Capucine Picard, Anne Durandy, Alain Fischer, Sven Kracker

PAX7 expression defines germline stem cells in the adult testis

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Abstract

Spermatogenesis is a complex, multistep process that maintains male fertility and is sustained by rare germline stem cells. Spermatogenic progression begins with spermatogonia, populations of which express distinct markers. The identity of the spermatogonial stem cell population in the undisturbed testis is controversial due to a lack of reliable and specific markers. Here we identified the transcription factor PAX7 as a specific marker of a rare subpopulation of A_single spermatogonia in mice. PAX7⁺ cells were present in the testis at birth. Compared with the adult testis, PAX7⁺ cells constituted a much higher percentage of neonatal germ cells. Lineage tracing in healthy adult mice revealed that PAX7⁺ spermatogonia self-maintained and produced expanding clones that gave rise to mature spermatozoa. Interestingly, in mice subjected to chemotherapy and radiotherapy, both of which damage the vast majority of germ cells and can result in sterility, PAX7⁺ spermatogonia selectively survived, and their subsequent expansion contributed to the recovery of spermatogenesis. Finally, PAX7⁺ spermatogonia were present in the testes of a diverse set of mammals. Our data indicate that the PAX7⁺ subset of A_single spermatogonia functions as robust testis stem cells that maintain fertility in normal spermatogenesis in healthy mice and mediate recovery after severe germline injury, such as occurs after cancer therapy.

Authors

Gina M. Aloisio, Yuji Nakada, Hatice D. Saatcioglu, Christopher G. Peña, Michael D. Baker, Edward D. Tarnawa, Jishnu Mukherjee, Hema Manjunath, Abhijit Bugde, Anita L. Sengupta, James F. Amatruda, Ileana Cuevas, F. Kent Hamra, Diego H. Castrillon

Shifting FcγRIIA-ITAM from activation to inhibitory configuration ameliorates arthritis

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Abstract

Rheumatoid arthritis–associated (RA-associated) inflammation is mediated through the interaction between RA IgG immune complexes and IgG Fc receptors on immune cells. Polymorphisms within the gene encoding the human IgG Fc receptor IIA (hFcγRIIA) are associated with an increased risk of developing RA. Within the hFcγRIIA intracytoplasmic domain, there are 2 conserved tyrosine residues arranged in a noncanonical immunoreceptor tyrosine–based activation motif (ITAM). Here, we reveal that inhibitory engagement of the hFcγRIIA ITAM either with anti-hFcγRII F(ab′)₂ fragments or intravenous hIgG (IVIg) ameliorates RA-associated inflammation, and this effect was characteristic of previously described inhibitory ITAM (ITAMi) signaling for hFcαRI and hFcγRIIIA, but only involves a single tyrosine. In hFcγRIIA-expressing mice, arthritis induction was inhibited following hFcγRIIA engagement. Moreover, hFcγRIIA ITAMi-signaling reduced ROS and inflammatory cytokine production through inhibition of guanine nucleotide exchange factor VAV-1 and IL-1 receptor–associated kinase 1 (IRAK-1), respectively. ITAMi signaling was mediated by tyrosine 304 (Y304) within the hFcγRIIA ITAM, which was required for recruitment of tyrosine kinase SYK and tyrosine phosphatase SHP-1. Anti-hFcγRII F(ab′)₂ treatment of inflammatory synovial cells from RA patients inhibited ROS production through induction of ITAMi signaling. These data suggest that shifting constitutive hFcγRIIA-mediated activation to ITAMi signaling could ameliorate RA-associated inflammation.

Authors

Sanae Ben Mkaddem, Gilles Hayem, Friederike Jönsson, Elisabetta Rossato, Erwan Boedec, Tarek Boussetta, Jamel El Benna, Pierre Launay, Jean-Michel Goujon, Marc Benhamou, Pierre Bruhns, Renato C. Monteiro

Lymphatic regulator PROX1 determines Schlemm’s canal integrity and identity

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Abstract

Schlemm’s canal (SC) is a specialized vascular structure in the eye that functions to drain aqueous humor from the intraocular chamber into systemic circulation. Dysfunction of SC has been proposed to underlie increased aqueous humor outflow (AHO) resistance, which leads to elevated ocular pressure, a factor for glaucoma development in humans. Here, using lymphatic and blood vasculature reporter mice, we determined that SC, which originates from blood vessels during the postnatal period, acquires lymphatic identity through upregulation of prospero homeobox protein 1 (PROX1), the master regulator of lymphatic development. SC expressed lymphatic valve markers FOXC2 and integrin α₉ and exhibited continuous vascular endothelial–cadherin (VE-cadherin) junctions and basement membrane, similar to collecting lymphatics. SC notably lacked luminal valves and expression of the lymphatic endothelial cell markers podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Using an ocular puncture model, we determined that reduced AHO altered the fate of SC both during development and under pathologic conditions; however, alteration of VEGF-C/VEGFR3 signaling did not modulate SC integrity and identity. Intriguingly, PROX1 expression levels linearly correlated with SC functionality. For example, PROX1 expression was reduced or undetectable under pathogenic conditions and in deteriorated SCs. Collectively, our data indicate that PROX1 is an accurate and reliable biosensor of SC integrity and identity.

Authors

Dae-Young Park, Junyeop Lee, Intae Park, Dongwon Choi, Sunju Lee, Sukhyun Song, Yoonha Hwang, Ki Yong Hong, Yoshikazu Nakaoka, Taija Makinen, Pilhan Kim, Kari Alitalo, Young-Kwon Hong, Gou Young Koh

The Schlemm’s canal is a VEGF-C/VEGFR-3–responsive lymphatic-like vessel

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Abstract

In glaucoma, aqueous outflow into the Schlemm’s canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.

Authors

Aleksanteri Aspelund, Tuomas Tammela, Salli Antila, Harri Nurmi, Veli-Matti Leppänen, Georgia Zarkada, Lukas Stanczuk, Mathias Francois, Taija Mäkinen, Pipsa Saharinen, Ilkka Immonen, Kari Alitalo

Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD

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Abstract

The pathogenesis of chronic obstructive pulmonary disease (COPD) remains unclear, but involves loss of alveolar surface area (emphysema) and airway inflammation (bronchitis) as the consequence of cigarette smoke (CS) exposure. Previously, we demonstrated that autophagy proteins promote lung epithelial cell death, airway dysfunction, and emphysema in response to CS; however, the underlying mechanisms have yet to be elucidated. Here, using cultured pulmonary epithelial cells and murine models, we demonstrated that CS causes mitochondrial dysfunction that is associated with a reduction of mitochondrial membrane potential. CS induced mitophagy, the autophagy-dependent elimination of mitochondria, through stabilization of the mitophagy regulator PINK1. CS caused cell death, which was reduced by administration of necrosis or necroptosis inhibitors. Genetic deficiency of PINK1 and the mitochondrial division/mitophagy inhibitor Mdivi-1 protected against CS-induced cell death and mitochondrial dysfunction in vitro and reduced the phosphorylation of MLKL, a substrate for RIP3 in the necroptosis pathway. Moreover, Pink1^–/– mice were protected against mitochondrial dysfunction, airspace enlargement, and mucociliary clearance (MCC) disruption during CS exposure. Mdivi-1 treatment also ameliorated CS-induced MCC disruption in CS-exposed mice. In human COPD, lung epithelial cells displayed increased expression of PINK1 and RIP3. These findings implicate mitophagy-dependent necroptosis in lung emphysematous changes in response to CS exposure, suggesting that this pathway is a therapeutic target for COPD.

Authors

Kenji Mizumura, Suzanne M. Cloonan, Kiichi Nakahira, Abhiram R. Bhashyam, Morgan Cervo, Tohru Kitada, Kimberly Glass, Caroline A. Owen, Ashfaq Mahmood, George R. Washko, Shu Hashimoto, Stefan W. Ryter, Augustine M.K. Choi

p38 signaling inhibits mTORC1-independent autophagy in senescent human CD8⁺ T cells

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Abstract

T cell senescence is thought to contribute to immune function decline, but the pathways that mediate senescence in these cells are not clear. Here, we evaluated T cell populations from healthy volunteers and determined that human CD8⁺ effector memory T cells that reexpress the naive T cell marker CD45RA have many characteristics of cellular senescence, including decreased proliferation, defective mitochondrial function, and elevated levels of both ROS and p38 MAPK. Despite their apparent senescent state, we determined that these cells secreted high levels of both TNF-α and IFN-γ and showed potent cytotoxic activity. We found that the senescent CD45RA-expressing population engaged anaerobic glycolysis to generate energy for effector functions. Furthermore, inhibition of p38 MAPK signaling in senescent CD8⁺ T cells increased their proliferation, telomerase activity, mitochondrial biogenesis, and fitness; however, the extra energy required for these processes did not arise from increased glucose uptake or oxidative phosphorylation. Instead, p38 MAPK blockade in these senescent cells induced an increase in autophagy through enhanced interactions between p38 interacting protein (p38IP) and autophagy protein 9 (ATG9) in an mTOR-independent manner. Together, our findings describe fundamental metabolic requirements of senescent primary human CD8⁺ T cells and demonstrate that p38 MAPK blockade reverses senescence via an mTOR-independent pathway.

Authors

Sian M. Henson, Alessio Lanna, Natalie E. Riddell, Ornella Franzese, Richard Macaulay, Stephen J. Griffiths, Daniel J. Puleston, Alexander Scarth Watson, Anna Katharina Simon, Sharon A. Tooze, Arne N. Akbar

PPARγ ablation sensitizes proopiomelanocortin neurons to leptin during high-fat feeding

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Abstract

Activation of central PPARγ promotes food intake and body weight gain; however, the identity of the neurons that express PPARγ and mediate the effect of this nuclear receptor on energy homeostasis is unknown. Here, we determined that selective ablation of PPARγ in murine proopiomelanocortin (POMC) neurons decreases peroxisome density, elevates reactive oxygen species, and induces leptin sensitivity in these neurons. Furthermore, ablation of PPARγ in POMC neurons preserved the interaction between mitochondria and the endoplasmic reticulum, which is dysregulated by HFD. Compared with control animals, mice lacking PPARγ in POMC neurons had increased energy expenditure and locomotor activity; reduced body weight, fat mass, and food intake; and improved glucose metabolism when exposed to high-fat diet (HFD). Finally, peripheral administration of either a PPARγ activator or inhibitor failed to affect food intake of mice with POMC-specific PPARγ ablation. Taken together, our data indicate that PPARγ mediates cellular, biological, and functional adaptations of POMC neurons to HFD, thereby regulating whole-body energy balance.

Authors

Lihong Long, Chitoku Toda, Jing Kwon Jeong, Tamas L. Horvath, Sabrina Diano

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance

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Abstract

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5–6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase–deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.

Authors

Felicity Payne, Rita Colnaghi, Nuno Rocha, Asha Seth, Julie Harris, Gillian Carpenter, William E. Bottomley, Eleanor Wheeler, Stephen Wong, Vladimir Saudek, David Savage, Stephen O’Rahilly, Jean-Claude Carel, Inês Barroso, Mark O’Driscoll, Robert Semple

Endothelial C-type natriuretic peptide maintains vascular homeostasis

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Abstract

The endothelium plays a fundamental role in maintaining vascular homeostasis by releasing factors that regulate local blood flow, systemic blood pressure, and the reactivity of leukocytes and platelets. Accordingly, endothelial dysfunction underpins many cardiovascular diseases, including hypertension, myocardial infarction, and stroke. Herein, we evaluated mice with endothelial-specific deletion of Nppc, which encodes C-type natriuretic peptide (CNP), and determined that this mediator is essential for multiple aspects of vascular regulation. Specifically, disruption of CNP leads to endothelial dysfunction, hypertension, atherogenesis, and aneurysm. Moreover, we identified natriuretic peptide receptor–C (NPR-C) as the cognate receptor that primarily underlies CNP-dependent vasoprotective functions and developed small-molecule NPR-C agonists to target this pathway. Administration of NPR-C agonists promotes a vasorelaxation of isolated resistance arteries and a reduction in blood pressure in wild-type animals that is diminished in mice lacking NPR-C. This work provides a mechanistic explanation for genome-wide association studies that have linked the NPR-C (Npr3) locus with hypertension by demonstrating the importance of CNP/NPR-C signaling in preserving vascular homoeostasis. Furthermore, these results suggest that the CNP/NPR-C pathway has potential as a disease-modifying therapeutic target for cardiovascular disorders.

Authors

Amie J. Moyes, Rayomand S. Khambata, Inmaculada Villar, Kristen J. Bubb, Reshma S. Baliga, Natalie G. Lumsden, Fang Xiao, Paul J. Gane, Anne-Sophie Rebstock, Roberta J. Worthington, Michela I. Simone, Filipa Mota, Fernando Rivilla, Susana Vallejo, Concepción Peiró, Carlos F. Sánchez Ferrer, Snezana Djordjevic, Mark J. Caulfield, Raymond J. MacAllister, David L. Selwood, Amrita Ahluwalia, Adrian J. Hobbs

Development and translational imaging of a TP53 porcine tumorigenesis model

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Abstract

Cancer is the second deadliest disease in the United States, necessitating improvements in tumor diagnosis and treatment. Current model systems of cancer are informative, but translating promising imaging approaches and therapies to clinical practice has been challenging. In particular, the lack of a large-animal model that accurately mimics human cancer has been a major barrier to the development of effective diagnostic tools along with surgical and therapeutic interventions. Here, we developed a genetically modified porcine model of cancer in which animals express a mutation in TP53 (which encodes p53) that is orthologous to one commonly found in humans (R175H in people, R167H in pigs). TP53^R167H/R167H mutant pigs primarily developed lymphomas and osteogenic tumors, recapitulating the tumor types observed in mice and humans expressing orthologous TP53 mutant alleles. CT and MRI imaging data effectively detected developing tumors, which were validated by histopathological evaluation after necropsy. Molecular genetic analyses confirmed that these animals expressed the R167H mutant p53, and evaluation of tumors revealed characteristic chromosomal instability. Together, these results demonstrated that TP53^R167H/R167H pigs represent a large-animal tumor model that replicates the human condition. Our data further suggest that this model will be uniquely suited for developing clinically relevant, noninvasive imaging approaches to facilitate earlier detection, diagnosis, and treatment of human cancers.

Authors

Jessica C. Sieren, David K. Meyerholz, Xiao-Jun Wang, Bryan T. Davis, John D. Newell Jr., Emily Hammond, Judy A. Rohret, Frank A. Rohret, Jason T. Struzynski, J. Adam Goeken, Paul W. Naumann, Mariah R. Leidinger, Agshin Taghiyev, Richard Van Rheeden, Jussara Hagen, Benjamin W. Darbro, Dawn E. Quelle, Christopher S. Rogers

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

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Abstract

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton’s tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2′-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro–B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

Authors

Burcu Bestas, Pedro M.D. Moreno, K. Emelie M. Blomberg, Dara K. Mohammad, Amer F. Saleh, Tolga Sutlu, Joel Z. Nordin, Peter Guterstam, Manuela O. Gustafsson, Shabnam Kharazi, Barbara Piątosa, Thomas C. Roberts, Mark A. Behlke, Matthew J.A. Wood, Michael J. Gait, Karin E. Lundin, Samir El Andaloussi, Robert Månsson, Anna Berglöf, Jesper Wengel, C.I. Edvard Smith

Gene therapy enhances chemotherapy tolerance and efficacy in glioblastoma patients

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Abstract

BACKGROUND. Temozolomide (TMZ) is one of the most potent chemotherapy agents for the treatment of glioblastoma. Unfortunately, almost half of glioblastoma tumors are TMZ resistant due to overexpression of methylguanine methyltransferase (MGMT^hi). Coadministration of O⁶-benzylguanine (O⁶BG) can restore TMZ sensitivity, but causes off-target myelosuppression. Here, we conducted a prospective clinical trial to test whether gene therapy to confer O⁶BG resistance in hematopoietic stem cells (HSCs) improves chemotherapy tolerance and outcome.

METHODS. We enrolled 7 newly diagnosed glioblastoma patients with MGMT^hi tumors. Patients received autologous gene-modified HSCs following single-agent carmustine administration. After hematopoietic recovery, patients underwent O⁶BG/TMZ chemotherapy in 28-day cycles. Serial blood samples and tumor images were collected throughout the study. Chemotherapy tolerance was determined by the observed myelosuppression and recovery following each cycle. Patient-specific biomathematical modeling of tumor growth was performed. Progression-free survival (PFS) and overall survival (OS) were also evaluated.

RESULTS. Gene therapy permitted a significant increase in the mean number of tolerated O⁶BG/TMZ cycles (4.4 cycles per patient, P < 0.05) compared with historical controls without gene therapy (n = 7 patients, 1.7 cycles per patient). One patient tolerated an unprecedented 9 cycles and demonstrated long-term PFS without additional therapy. Overall, we observed a median PFS of 9 (range 3.5–57⁺) months and OS of 20 (range 13–57⁺) months. Furthermore, biomathematical modeling revealed markedly delayed tumor growth at lower cumulative TMZ doses in study patients compared with patients that received standard TMZ regimens without O⁶BG.

CONCLUSION. These data support further development of chemoprotective gene therapy in combination with O⁶BG and TMZ for the treatment of glioblastoma and potentially other tumors with overexpression of MGMT.

TRIAL REGISTRATION. Clinicaltrials.gov NCT00669669.

FUNDING. R01CA114218, R01AI080326, R01HL098489, P30DK056465, K01DK076973, R01HL074162, R01CA164371, R01NS060752, U54CA143970.

Authors

Jennifer E. Adair, Sandra K. Johnston, Maciej M. Mrugala, Brian C. Beard, Laura A. Guyman, Anne L. Baldock, Carly A. Bridge, Andrea Hawkins-Daarud, Jennifer L. Gori, Donald E. Born, Luis F. Gonzalez-Cuyar, Daniel L. Silbergeld, Russell C. Rockne, Barry E. Storer, Jason K. Rockhill, Kristin R. Swanson, Hans-Peter Kiem

Menin determines K-RAS proliferative outputs in endocrine cells

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Abstract

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic β cells for people with diabetes and for targeting menin-sensitive endocrine tumors.

Authors

Chester E. Chamberlain, David W. Scheel, Kathleen McGlynn, Hail Kim, Takeshi Miyatsuka, Juehu Wang, Vinh Nguyen, Shuhong Zhao, Anastasia Mavropoulos, Aswin G. Abraham, Eric O’Neill, Gregory M. Ku, Melanie H. Cobb, Gail R. Martin, Michael S. German

A selective microRNA-based strategy inhibits restenosis while preserving endothelial function

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Abstract

Drugs currently approved to coat stents used in percutaneous coronary interventions do not discriminate between proliferating vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). This lack of discrimination delays reendothelialization and vascular healing, increasing the risk of late thrombosis following angioplasty. We developed a microRNA-based (miRNA-based) approach to inhibit proliferative VSMCs, thus preventing restenosis, while selectively promoting reendothelialization and preserving EC function. We used an adenoviral (Ad) vector that encodes cyclin-dependent kinase inhibitor p27^Kip1 (p27) with target sequences for EC-specific miR-126-3p at the 3′ end (Ad-p27-126TS). Exogenous p27 overexpression was evaluated in vitro and in a rat arterial balloon injury model following transduction with Ad-p27-126TS, Ad-p27 (without miR-126 target sequences), or Ad-GFP (control). In vitro, Ad-p27-126TS protected the ability of ECs to proliferate, migrate, and form networks. At 2 and 4 weeks after injury, Ad-p27-126TS–treated animals exhibited reduced restenosis, complete reendothelialization, reduced hypercoagulability, and restoration of the vasodilatory response to acetylcholine to levels comparable to those in uninjured vessels. By incorporating miR-126-3p target sequences to leverage endogenous EC-specific miR-126, we overexpressed exogenous p27 in VSMCs, while selectively inhibiting p27 overexpression in ECs. Our proof-of-principle study demonstrates the potential of using a miRNA-based strategy as a therapeutic approach to specifically inhibit vascular restenosis while preserving EC function.

Authors

Gaetano Santulli, Anetta Wronska, Kunihiro Uryu, Thomas G. Diacovo, Melanie Gao, Steven O. Marx, Jan Kitajewski, Jamie M. Chilton, Kemal Marc Akat, Thomas Tuschl, Andrew R. Marks, Hana Totary-Jain

Early efficacy trial of anakinra in corticosteroid-resistant autoimmune inner ear disease

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Abstract

BACKGROUND. Autoimmune inner ear disease (AIED) is a rare disease that results in progressive sensorineural hearing loss. Patients with AIED initially respond to corticosteroids; however, many patients become unresponsive to this treatment over time, and there is no effective alternative therapy for these individuals.

METHODS. We performed a phase I/II open-label, single-arm clinical trial of the IL-1 receptor antagonist anakinra in corticosteroid-resistant AIED patients. Given that the etiology of corticosteroid resistance is likely heterogeneous, we used a Simon 2-stage design to distinguish between an unacceptable (≤10%) and an acceptable (≥30%) response rate to anakinra therapy. Subjects received 100 mg anakinra by subcutaneous injection for 84 days, followed by a 180-day observational period.

RESULTS. Based on patient responses, the Simon 2-stage rule permitted premature termination of the trial after 10 subjects completed the 84-day drug period, as the target efficacy for the entire trial had been achieved. Of these 10 patients, 7 demonstrated audiometric improvement, as assessed by pure tone average (PTA) and word recognition score (WRS). In these 7 responders, reduced IL-1β plasma levels correlated with clinical response. Upon discontinuation of treatment, 3 subjects relapsed, which correlated with increased IL-1β plasma levels.

CONCLUSION. We demonstrated that IL-1β inhibition in corticosteroid-resistant AIED patients was effective in a small cohort of patients and that IL-1β plasma levels associated with both clinical hearing response and disease relapse. These results suggest that a larger phase II randomized clinical trial of IL-1β inhibition is warranted.

TRIAL REGISTRATION. ClinicalTrials.gov NCT01267994.

FUNDING. NIH, Merrill & Phoebe Goodman Otology Research Center, and Long Island Hearing & Speech Society.

Authors

Andrea Vambutas, Martin Lesser, Virginia Mullooly, Shresh Pathak, Gerald Zahtz, Lisa Rosen, Elliot Goldofsky

Sexually dimorphic RB inactivation underlies mesenchymal glioblastoma prevalence in males

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Abstract

The prevalence of brain tumors in males is common but unexplained. While sex differences in disease are typically mediated through acute sex hormone actions, sex-specific differences in brain tumor rates are comparable at all ages, suggesting that factors other than sex hormones underlie this discrepancy. We found that mesenchymal glioblastoma (Mes-GBM) affects more males as the result of cell-intrinsic sexual dimorphism in astrocyte transformation. We used astrocytes from neurofibromin-deficient (Nf1^–/–) mice expressing a dominant-negative form of the tumor suppressor p53 (DNp53) and treated them with EGF as a Mes-GBM model. Male Mes-GBM astrocytes exhibited greater growth and colony formation compared with female Mes-GBM astrocytes. Moreover, male Mes-GBM astrocytes underwent greater tumorigenesis in vivo, regardless of recipient mouse sex. Male Mes-GBM astrocytes exhibited greater inactivation of the tumor suppressor RB, higher proliferation rates, and greater induction of a clonogenic, stem-like cell population compared with female Mes-GBM astrocytes. Furthermore, complete inactivation of RB and p53 in Mes-GBM astrocytes resulted in equivalent male and female tumorigenic transformation, indicating that intrinsic differences in RB activation are responsible for the predominance of tumorigenic transformation in male astrocytes. Together, these results indicate that cell-intrinsic sex differences in RB regulation and stem-like cell function may underlie the predominance of GBM in males.

Authors

Tao Sun, Nicole M. Warrington, Jingqin Luo, Michael D. Brooks, Sonika Dahiya, Steven C. Snyder, Rajarshi Sengupta, Joshua B. Rubin

Mesenchymal high-grade glioma is maintained by the ID-RAP1 axis

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Abstract

Authors

Francesco Niola, Xudong Zhao, Devendra Singh, Ryan Sullivan, Angelica Castano, Antonio Verrico, Pietro Zoppoli, Dinorah Friedmann-Morvinski, Erik Sulman, Lindy Barrett, Yuan Zhuang, Inder Verma, Robert Benezra, Ken Aldape, Antonio Iavarone, Anna Lasorella