Filgotinib suppresses HIV-1-driven gene transcription by inhibiting HIV-1 splicing and T cell activation

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Abstract

Despite effective antiretroviral therapy, HIV-1-nfected cells continue to produce viral antigens and induce chronic immune exhaustion. We propose to identify HIV-1-suppressing agents which can inhibit HIV-1 reactivation and reduce HIV-1-induced immune activation. Using a novel dual reporter system and a high-throughput drug screen, we identified FDA-approved drugs which can suppress HIV-1 reactivation in both cell line models and CD4+ T cells from virally suppressed, HIV-1-infected individuals. We identified 11 cellular pathways required for HIV-1 reactivation as druggable targets. Using differential expression analysis, gene set enrichment analysis and exon-intron landscape analysis, we examined the impact of drug treatment on the cellular environment at a genome-wide level. We identified a new function of a JAK inhibitor filgotinib which suppresses HIV-1 splicing. First, filgotinib preferentially suppresses spliced HIV-1 RNA transcription. Second, filgotinib suppresses HIV-1-driven aberrant cancer-related gene expression at the integration site. Third, we found that filgotinib suppresses HIV-1 transcription by inhibiting T cell activation and by modulating RNA splicing. Finally, we found that filgotinib treatment reduces the proliferation of HIV-1-infected cells. Overall, the combination of a drug screen and transcriptome analysis provides systemic understanding of cellular targets required for HIV-1 reactivation and drug candidates that may reduce HIV-1-related immune activation.

Authors

Yang-Hui Jimmy Yeh, Katharine M. Jenike, Rachela M. Calvi, Jennifer Chiarella, Rebecca Hoh, Steven G. Deeks, Ya-Chi Ho

FGFR4 regulates tumor subtype differentiation in luminal breast cancer and metastatic disease

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Abstract

Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of Luminal A primary breast tumors to give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2-). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis FGFR4 likely participates in this subtype switching. To evaluate this, we developed two FGFR4 genomic signatures using a PDX model treated with a FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single cell RNAseq demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the METABRIC breast cancer cohort,FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, FGFR4-induced signature was also an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastasis revealed that the FGFR4-induced signature was significantly higher in luminal/ER+ tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification.

Authors

Susana Garcia-Recio, Aatish Thennavan, Michael P. East, Joel S. Parker, Juan M. Cejalvo, Joseph P. Garay, Daniel P. Hollern, Xiaping He, Kevin R. Mott, Patricia Galván, Cheng Fan, Sara R. Selitsky, Alisha R. Coffey, David Marron, Fara Brasó-Maristany, Octavio Burgues, Joan Albanell, Federico Rojo, Ana Lluch, Eduardo Martinez de Dueñas, Jeffrey M. Rosen, Gary L. Johnson, Lisa A. Carey, Aleix Prat, Charles M. Perou

p38 mitogen-activated protein kinase regulates chamber specific perinatal growth in heart

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Abstract

In mammalian heart, left ventricle (LV) rapidly becomes more dominant in size and function over right ventricle (RV) after birth. The molecular regulators responsible for this chamber specific differential growth are largely unknown. We found the cardiomyocytes in neonatal mouse RV had lower proliferation, more apoptosis and smaller sizes comparing to the LV. Such chamber specific growth pattern was associated with a selective activation of p38 MAPK activity in the RV and simultaneous inactivation in the LV. Cardiomyocyte-specific deletion of both mapk14 and mapk11 genes in mice results in loss of p38 MAP kinase expression and activity in the neonatal heart. Inactivation of p38 activity led to marked increase in myocytes proliferation and hypertrophy but diminished myocyte apoptosis, specifically in the RV. Consequently, the p38 inactivated hearts showed RV specific enlargement postnatally, progressing to pulmonary hypertension and right heart failure at adult stage. Chamber-specific p38 activity was associated with differential expression of dual-specific phosphatases (DUSPs) in neonatal hearts, including Dusp26. Unbiased transcriptome analysis revealed IRE1/XBP mediated gene regulation contributed to p38 MAPK dependent regulation of neonatal myocyte proliferation and binucleation. These findings establish an obligatory role of DUSP-p38-IRE1 signaling in myocytes for chamber specific growth in postnatal heart.

Authors

Tomohiro Yokota, Jin LI, Jijun Huang, Zhaojun Xiong, Qing Zhang, Tracey W. Chan, Yichen Ding, Christoph D. Rau, Kevin Sung, Shuxun Ren, Rajan P. Kulkarni, Tzung Hsiai, Xinshu Xiao, Marlin Touma, Susumu Minamisawa, Yibin Wang

Anti-retroviral therapy does not reduce tuberculosis reactivation in a tuberculosis-HIV co-infection model

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Abstract

While the advent of combination antiretroviral therapy (ART) has significantly improved survival, tuberculosis (TB) remains the leading cause of death in the HIV-infected population. We employed Mtb/Simian Immunodeficiency Virus (SIV) co-infected macaques to model Mtb/HIV co-infection and study the impact of ART on TB reactivation due to HIV-infection. While ART significantly reduced viral loads and increased CD4+ T cell counts in whole blood and BAL samples, it did not reduce the relative risk of SIV- induced TB reactivation in ART treated macaques in the early phase of treatment. CD4+ T cells were poorly restored specifically in the lung interstitium, despite their significant restoration in the alveolar compartment of the lung as well as in the periphery. IDO1 induction on myeloid cells in the iBALT likely contributed to dysregulated T cell homing and impaired lung immunity. Thus, while ART is indispensable in controlling viral replication, CD4+ T cells restoration and preventing opportunistic infection, it appears inadequate in reversing clinical signs of TB reactivation during the relatively short duration of ART and follow-up during this study. This warrants modeling concurrent treatment of TB and HIV to potentially reduce the risk of reactivation of TB due to HIV. The current and future studies like this have the potential to inform treatment strategies in patients with Mtb/HIV co-infection.

Authors

Shashank R. Ganatra, Allison N. Bucsan, Xavier Alvarez, Shyamesh Kumar, Ayan Chatterjee, Melanie Quezada, Abigail I. Fish, Dhiraj K. Singh, Bindu Singh, Riti Sharan, Tae-Hyung Lee, Uma Shanmugasundaram, Vijayakumar Velu, Shabaana A. Khader, Smriti Mehra, Jyothi Rengarajan, Deepak Kaushal

Tau is not necessary for amyloid-beta-induced synaptic and memory impairments

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Abstract

The amyloid hypothesis posits that the amyloid-beta (Aβ) protein precedes and requires microtubule-associated protein tau in a sort of trigger-bullet mechanism leading to Alzheimer’s disease (AD) pathology. This sequence of events has become dogmatic in the AD field and is used to explain clinical trial failures due to a late start of the intervention when Aβ already activated tau. Here, using a multidisplinary approach combining molecular biological, biochemical, histopathological, electrophysiological and behavioral methods we demonstrated that tau suppression did not protect against Aβ-induced damage of long-term synaptic plasticity and memory, as well as amyloid deposition. Tau suppression could even unravel a defect in basal synaptic transmission in a mouse model of amyloid deposition. Similarly, tau suppression did not protect against exogenous oligomeric tau induced impairment of long-term synaptic plasticity and memory. The protective effect of tau suppression was, in turn, confined to short-term plasticity and memory. Taken together, our data suggest that therapies downstream of Aβ and tau together are more suitable to combat AD than therapies against one or the other alone.

Authors

Daniela Puzzo, Elentina K. Argyrousi, Agnieszka Staniszewski, Hong Zhang, Elisa Calcagno, Elisa Zuccarello, Erica Acquarone, Mauro Fà, Domenica Donatella Li Puma, Claudio Grassi, Luciano D'Adamio, Nicholas M. Kanaan, Paul E. Fraser, Ottavio Arancio

3D model of harlequin ichthyosis reveals inflammatory therapeutic targets

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Abstract

The biology of harlequin ichthyosis (HI), a devastating skin disorder, caused by loss of function mutations in the gene ABCA12, is poorly understood and to date no satisfactory treatment has been developed. We sought to investigate pathomechanisms of HI which could lead to the identification of new treatments to improve patients’ quality of life. In this study, RNA-Seq and functional assays were performed to define the effects of loss of ABCA12, using HI patient skin samples and an engineered CRISPR-Cas9 ABCA12 KO cell line. The HI living skin equivalent (3D model) recapitulated the HI skin phenotype. The cytokines IL-36α and IL-36γ were upregulated in HI skin whereas the innate immune inhibitor, IL-37, was strongly downregulated. We also identified STAT1 and its downstream target inducible nitric oxide synthase (NOS2) to be upregulated in the in vitro HI 3D model and HI patient skin samples. Inhibition of NOS2 using the inhibitor, 1400W, or the JAK inhibitor, tofacitinib, dramatically improved the in vitro HI phenotype by restoring the lipid barrier in the HI 3D model. Our study has identified dysregulated pathways in HI skin that are feasible therapeutic targets.

Authors

Florence Enjalbert, Priya Dewan, Matthew P. Caley, Eleri M. Jones, Mary A. Morse, David P. Kelsell, Anton J. Enright, Edel A. O'Toole

Histone methyltransferase MLL4 controls myofiber identity and muscle performance through MEF2 interaction

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Abstract

Skeletal muscle depends on the precise orchestration of contractile and metabolic gene expression programs to direct fiber type specification and to ensure muscle performance. Exactly how such fiber type-specific patterns of gene expression are established and maintained remains unclear, however. Here, we demonstrate that histone mono-methyltransferase MLL4 (KMT2D), an enhancer regulator enriched in slow myofibers, plays a critical role in controlling muscle fiber identity as well as muscle performance. Skeletal muscle-specific ablation of MLL4 in mice resulted in downregulation of the slow-oxidative myofiber gene program, decreased number of type I myofibers, and diminished mitochondrial respiration, which caused reductions in muscle fat utilization and endurance capacity during exercise. Genome-wide ChIP-seq and mRNA-seq analyses revealed that MLL4 directly binds to enhancers and functions as a coactivator of the myocyte enhancer factor 2 (MEF2) to activate transcription of slow-oxidative myofiber genes. Importantly, we also found that the MLL4 regulatory circuit is associated with muscle fiber type remodeling in humans. Thus, our results uncover a pivotal role for MLL4 in specifying structural and metabolic identities of myofibers that govern muscle performance. These findings provide new therapeutic opportunities for enhancing muscle fitness to combat a variety of metabolic and muscular diseases.

Authors

Lin Liu, Chenyun Ding, Tingting Fu, Zhenhua Feng, Ji-Eun Lee, Liwei Xiao, Zhisheng Xu, Yujing Yin, Qiqi Guo, Zongchao Sun, Wanping Sun, Yan Mao, Likun Yang, Zheng Zhou, Danxia Zhou, Leilei Xu, Zezhang Zhu, Yong Qiu, Kai Ge, Zhenji Gan

Impact of TREM2^R47H variant on tau pathology-induced gliosis and neurodegeneration

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Abstract

Alzheimer’s disease (AD) is characterized by amyloid-β-containing plaques and neurofibrillary tangles composed of aggregated, hyperphosphorylated tau. Beyond tau and Aβ, evidence suggests that microglia play an important role in AD pathogenesis. Rare variants in the microglial-expressed triggering receptor expressed on myeloid cells 2 (TREM2) gene increase AD risk 2-4-fold. It is likely that these TREM2 variants increase AD risk by decreasing the response of microglia to Aβ and its local toxicity. However, neocortical Aβ pathology occurs many years before neocortical tau pathology in AD. Thus, it will be important to understand the role of TREM2 in the context of tauopathy. We investigated the impact of the AD-associated TREM2 variant (R47H) on tau-mediated neuropathology in the PS19 mouse model of tauopathy. We assessed PS19 mice expressing human TREM2CV (common variant) or human TREM2R47H. PS19-T2R47H mice had significantly attenuated brain atrophy and synapse loss versus PS19-T2CV mice. Gene expression analyses and CD68 immunostaining revealed attenuated microglial reactivity in PS19-T2R47H versus PS19-T2CV mice. There was also a decrease in phagocytosis of postsynaptic elements by microglia expressing TREM2R47H in the PS19 mice and in human AD brains. These findings suggest that impaired TREM2 signaling reduces microglia-mediated neurodegeneration in the setting of tauopathy.

Authors

Maud Gratuze, Cheryl E.G. Leyns, Andrew D. Sauerbeck, Marie-Kim St-Pierre, Monica Xiong, Nayeon Kim, Javier Remolina Serrano, Marie-Ève Tremblay, Terrance T. Kummer, Marco Colonna, Jason D. Ulrich, David M. Holtzman

Melanocortin-4 receptor antagonist TCMCB07 ameliorates cancer- and chronic kidney disease-associated cachexia

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Abstract

Cachexia, a devastating wasting syndrome characterized by severe weight loss with specific losses of muscle and adipose tissue, is driven by reduced food intake, increased energy expenditure, excess catabolism, and inflammation. Cachexia is associated with poor prognosis and high mortality, and frequently occurs in patients with cancer, chronic kidney disease, infection, and many other illnesses. There is no effective treatment for this condition. Hypothalamic melanocortins have a potent and long-lasting inhibitory effect on feeding and anabolism, and pathophysiological processes increase melanocortin signaling tone leading to anorexia, metabolic changes, and eventual cachexia. We utilized three rat models of anorexia and cachexia (LPS, methylcholanthrene sarcoma, and 5/6 subtotal nephrectomy) to evaluate efficacy of TCMCB07, a synthetic antagonist of the melanocortin-4 receptor. Our data show that peripheral treatment of TCMCB07 with intraperitoneal, subcutaneous, and oral administration increased food intake and body weight, and preserved fat mass and lean mass during cachexia and LPS-induced anorexia. Furthermore, administration of TCMCB07 diminished hypothalamic inflammatory gene expression in cancer cachexia. These results suggest that peripheral TCMCB07 treatment effectively inhibits central melanocortin signaling and therefore stimulates appetite and enhances anabolism, indicating that TCMCB07 is a promising drug candidate to treat cachexia.

Authors

Xinxia Zhu, Michael F. Callahan, Kenneth A. Gruber, Marek Szumowski, Daniel L. Marks

Auxiliary trafficking subunit GJA1-20k protects Connexin43 from degradation and limits ventricular arrhythmias

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Abstract

Connexin 43 (Cx43) gap junctions provide intercellular coupling which ensures rapid action potential propagation and synchronized heart contraction. Altered Cx43 localization and reduced gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full length Cx43 but not GJA1-20k. We find that GJA1M213L/M213L mice have severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, reduced gap junctions, and die suddenly at two to four weeks of age. Heterozygous GJA1M213L/WT mice survive to adulthood with increased ventricular ectopy. Biochemical experiments indicate that cytoplasmic Cx43 has a half life that is 50% shorter than membrane associated Cx43. Without GJA1-20k, poorly trafficked Cx43 is degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart.

Authors

Shaohua Xiao, Daisuke Shimura, Rachel Baum, Diana M. Hernandez, Sosse Agvanian, Yoshiko Nagaoka, Makoto Katsumata, Paul D. Lampe, Andre G. Kleber, TingTing Hong, Robin M. Shaw