Idiopathic pulmonary fibrosis (IPF) is a chronic and deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) by myofibroblasts is a key factor that drives disease pathogenesis, although the underlying mechanisms remain unknown. Alternative polyadenylation (APA) has recently been shown to play a major role in cellular responses to stress by driving the expression of fibrotic factors and ECMs through altering microRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrate that CFIm25, a global regulator of APA, is down-regulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in alpha-smooth muscle actin (α-SMA) positive fibroblasts. Following the knockdown of CFIm25 in normal human lung fibroblasts, we identified 808 genes with shortened 3′UTRs, including those involved in the transforming growth factor-β signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key pro-fibrotic factors can be suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrate that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxd1 promoter enhances pulmonary fibrosis after bleomycin exposure in mice. Taken together, our results identified CFIm25 down-regulation as a novel mechanism to elevate pro-fibrotic gene expression in pulmonary fibrosis.
Tingting Weng, Junsuk Ko, Chioniso P. Masamha, Zheng Xia, Yu Xiang, Ning-yuan Chen, Jose G. Molina, Scott Collum, Tinne C. Mertens, Fayong Luo, Kemly Philip, Jonathan Davies, Jingjing Huang, Cory Wilson, Rajarajan A. Thandavarayan, Brian A. Bruckner, Soma S.K. Jyothula, Kelly A. Volcik, Lei Li, Leng Han, Wei Li, Shervin Assassi, Harry Karmouty-Quintana, Eric J. Wagner, Michael R. Blackburn
Bone osteogenic sarcoma has a poor prognosis as the exact cell of origin and the signaling pathways underling tumor formation remain undefined. Here, we report an osteogenic tumor mouse model based on the conditional knockout of liver kinase b1 (Lkb1; also known as Stk11) in Cathepsin K (Ctsk)-Cre expressing cells. Lineage tracing studies demonstrated that Ctsk-Cre could label a population of periosteal cells. The cells functioned as mesenchymal progenitors with regard to markers and functional properties. LKB1 deficiency increased proliferation and osteoblast differentiation of Ctsk+ periosteal cells, while downregulation of mTORC1 activity, using Raptor genetic mouse model or mTORC1 inhibitor treatment, ameliorated tumor progression of Ctsk-Cre Lkb1fllfl mice. Xenograft mouse models, using human osteosarcoma cell lines, also demonstrated that LKB1 deficiency promoted tumor formation, while mTOR inhibition suppressed xenograft tumor growth. In summary, we identified periosteum-derived Ctsk-Cre expressing cells as a cell of origin for osteogenic tumor and suggested the LKB1-mTORC1 pathway as a promising target for treatment of osteogenic tumor.
Yujiao Han, Heng Feng, Jun Sun, Xiaoting Liang, Zhuo Wang, Wenhui Xing, Qinggang Dai, Yang Yang, Anjia Han, Zhanying Wei, Qing Bi, Hongbin Ji, Tiebang Kang, Weiguo Zou
Non-apoptotic forms of cell death can trigger sterile inflammation through the release of danger-associated molecular patterns, which are recognized by innate immune receptors. However, despite years of investigation the mechanisms which initiate inflammatory responses after heart transplantation remain elusive. Here, we demonstrate that ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, decreases the level of pro-ferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamine, reduces cardiomyocyte cell death and blocks neutrophil recruitment following heart transplantation. Inhibition of necroptosis had no effect on neutrophil trafficking in cardiac grafts. We extend these observations to a model of coronary artery ligation-induced myocardial ischemia reperfusion injury where inhibition of ferroptosis resulted in reduced infarct size, improved left ventricular systolic function, and reduced left ventricular remodeling. Using intravital imaging of cardiac transplants, we uncover that ferroptosis orchestrates neutrophil recruitment to injured myocardium by promoting adhesion of neutrophils to coronary vascular endothelial cells through a TLR4/TRIF/type I IFN signaling pathway. Thus, we have discovered that inflammatory responses after cardiac transplantation are initiated through ferroptotic cell death and TLR4/Trif-dependent signaling in graft endothelial cells. These findings provide a platform for the development of therapeutic strategies for heart transplant recipients and patients, who are vulnerable to ischemia reperfusion injury following restoration of coronary blood flow.
Wenjun Li, Guoshuai Feng, Jason M. Gauthier, Inessa Lokshina, Ryuji Higashikubo, Sarah Evans, Xinping Liu, Adil Hassan, Satona Tanaka, Markus Cicka, Hsi-Min Hsiao, Daniel Ruiz-Perez, Andrea Bredemeyer, Richard W. Gross, Douglas L. Mann, Yulia Y. Tyurina, Andrew E. Gelman, Valerian E. Kagan, Andreas Linkermann, Kory J. Lavine, Daniel Kreisel
Anti-leukemic effect of BET/BRD4 (BETP) protein inhibition has been largely attributed to transcriptional downregulation of cellular anabolic/anti-apoptotic processes but its effect on bone marrow microenvironment, a sanctuary favoring persistence of leukemia stem/progenitor cells, is unexplored. Sustained degradation of BETP with small-molecule BET proteolysis-targeting chimera (PROTAC), ARV-825, resulted in marked downregulation of surface CXCR4 and CD44, key proteins in leukemia-microenvironment interaction, in AML cells. Abrogation of surface CXCR4 expression impaired SDF-1α directed migration and was mediated through transcriptional down-regulation of PIM1 kinase that in turn phosphorylates CXCR4 and facilitates its surface localization. Down-regulation of CD44/CD44v8-10 impaired cystine uptake, lowered intracellular reduced glutathione and increased oxidative stress. More importantly, BETP degradation markedly decreased CD34+CD38-CD90-CD45RA+ leukemic stem cell population and alone or in combination with Cytarabine, prolonged survival in mouse model of human leukemia including AML-PDX. Gene expression profiling and single cell proteomics confirmed down regulation of the gene signatures associated with ‘stemness’ in AML and Wnt/β-catenin, Myc pathways. Hence, BETP degradation by ARV-825 simultaneously targets cell intrinsic signaling, stromal interactions and metabolism in AML.
Sujan Piya, Hong Mu, Seemana Bhattacharya, Philip L. Lorenzi, R. Eric Davis, Teresa McQueen, Vivian Ruvolo, Natalia Baran, Zhiqiang Wang, Yimin Qian, Craig M. Crews, Marina Konopleava, Jo Ishizawa, M. James You, Hagop Kantarjian, Michael Andreeff, Gautam Borthakur
Gastrointestinal stromal tumor (GIST) is the most common human sarcoma, frequently characterized by an oncogenic mutation in the KIT or platelet-derived growth factor receptor alpha (PDGFRA) genes. We performed RNA sequencing of 75 human GIST tumors from 75 patients, comprising the largest cohort of GISTs sequenced to date, in order to discover differences in the immune infiltrates of KIT and PDGFRA-mutant GIST. Through bioinformatics, immunohistochemistry, and flow cytometry, we found that PDGFRA-mutant GISTs harbored more immune cells with increased cytolytic activity when compared to KIT-mutant GISTs. PDGFRA-mutant GISTs expressed many chemokines, such as CXCL14, at a significantly higher level when compared to KIT-mutant GISTs and exhibited more diverse driver-derived neoepitope:HLA binding, both of which may contribute to PDGFRA-mutant GIST immunogenicity. Through machine learning, we generated gene expression-based immune profiles capable of differentiating KIT and PDGFRA-mutant GISTs, and also identified additional immune features of high PD-1 and PD-L1 expressing tumors across all GIST mutational subtypes, which may provide insight into immunotherapeutic opportunities and limitations in GIST.
Gerardo A. Vitiello, Timothy G. Bowler, Mengyuan Liu, Benjamin D. Medina, Jennifer Q. Zhang, Nesteene J. Param, Jennifer K. Loo, Rachel L. Goldfeder, Frederic Chibon, Ferdinand Rossi, Shan Zeng, Ronald P. DeMatteo
Soluble urokinase receptor (suPAR) is a circulatory molecule that activates αvβ3 integrin on podocytes, causes foot process effacement, and contributes to proteinuric kidney disease. While active integrin can be targeted by antibodies and small molecules, endogenous inhibitors haven’t been discovered yet. Here we report a novel, renoprotective role for inducible costimulator (ICOS) ligand (ICOSL) in early kidney disease through its selective binding to podocyte αvβ3 integrin. Contrary to ICOSL’s immune-regulatory role, ICOSL in non-hematopoietic cells limited the activation of αvβ3 integrin. Specifically, ICOSL contains arginine-glycine-aspartate (RGD) motif, which allowed for a high affinity and selective binding to αvβ3 and modulation of podocyte adhesion. This binding was largely inhibited either by a synthetic RGD peptide or by a disrupted RGD sequence in ICOSL. ICOSL binding favored the active αvβ3 rather than the inactive form and showed little affinity for other integrins. Consistent with the rapid induction of podocyte ICOSL by inflammatory stimuli, glomerular ICOSL expression was increased in biopsies of early stage human proteinuric kidney diseases. Icosl deficiency in mice resulted in an increased susceptibility to proteinuria that was rescued by recombinant ICOSL. Our work identified a novel role for ICOSL, which serves as an endogenous αvβ3-selective antagonist to maintain glomerular filtration.
Kwi Hye Koh, Yanxia Cao, Steve Mangos, Nicholas J. Tardi, Ranadheer R. Dande, Ha Won Lee, Beata Samelko, Mehmet M. Altintas, Vincent P. Schmitz, Hyun Lee, Kamalika Mukherjee, Vasil Peev, David J. Cimbaluk, Jochen Reiser, Eunsil Hahm
Pancreatic ductal adenocarcinoma (PDAC) represents an immune quiescent tumor that is resistant to immune checkpoint inhibitors. Previously, our group has shown that a GM-CSF secreting allogenic pancreatic tumor cell vaccine (GVAX), may prime the tumor microenvironment by inducing intratumoral T-cell infiltration. Here, we show that untreated PDACs express minimal indoleamine-2, 3-dioxygenase (IDO1); however, GVAX therapy induced IDO1 expression on tumor epithelia as well as vaccine-induced tertiary lymphoid aggregates. IDO1 expression plays a role in regulating the polarization of Th1, Th17, and possibly T-regulatory cells in PDAC tumors. IDO1 inhibitor enhanced anti-tumor efficacy of GVAX in a murine model of PDACs. The combination of vaccine and IDO1 inhibitor enhanced intratumoral T-cell infiltration and function, but adding anti-PD-L1 antibody to the combination did not offer further synergy and in fact may have a negative interaction decreasing the number of intratumoral effector T-cells. Additionally, IDO1 inhibitor in the presence of vaccine therapy, did not significantly modulate intratumoral myeloid derived suppressor cells quantitatively, but diminished their suppressive effect on CD8+ proliferation. Our study thus supports the combination of IDO1 inhibitor and vaccine therapy, however, does not support the combination of IDO1 inhibitor and anti-PD-1/PD-L1 antibody for T cell-inflamed tumors such as PDACs treated with vaccine therapy.
Alex B. Blair, Jennifer Kleponis, Dwayne L. Thomas II, Stephen T. Muth, Adrian G. Murphy, Victoria Kim, Lei Zheng
Understanding the tumor immune microenvironment (TIME) promises to be key for optimal cancer therapy, especially in triple-negative breast cancer (TNBC). Integrating spatial resolution of immune cells with laser capture microdissection gene expression profiles, we defined distinct TIME stratification in TNBC with implications for current therapies, including immune checkpoint blockade. TNBCs with an immunoreactive microenvironment exhibited tumoral infiltration of granzyme B+ CD8+ T cells, a type I interferon signature, elevated expression of multiple immune inhibitory molecules, including IDO, PD-L1, and good outcome. An “immune-cold” microenvironment with absence of tumoral CD8+ T cells was defined by elevated expression of the immunosuppressive marker B7-H4, signatures of fibrotic stroma and poor outcome. A distinct poor outcome immunomodulatory microenvironment, hitherto poorly characterized, exhibited stromal restriction of CD8+ T cells, stromal expression of PD-L1 and enrichment for signatures of cholesterol biosynthesis. Metasignatures defining these TIME subtypes stratified TNBC, predicting outcome and identifying potential therapeutic targets for TNBC.
Tina Gruosso, Mathieu Gigoux, Venkata Satya Kumar Manem, Nicholas Bertos, Dongmei Zuo, Irina Perlitch, Sadiq Mehdi Ismail Saleh, Hong Zhao, Margarita Souleimanova, Radia Marie Johnson, Anne Monette, Valentina Munoz Ramos, Michael Trevor Hallett, John Stagg, Réjean Lapointe, Atilla Omeroglu, Sarkis Meterissian, Laurence Buisseret, Gert Van den Eynden, Roberto Salgado, Marie-Christine Guiot, Benjamin Haibe-Kains, Morag Park
Constitutive JAK2 signaling is central to myeloproliferative neoplasm (MPN) pathogenesis and results in activation of STAT, PI3K/AKT and MEK/ERK signaling. However, the therapeutic efficacy of current JAK2 inhibitors is limited. We investigated the role of MEK/ERK signaling in MPN cell survival in the setting of JAK kinase inhibition. Type I and II JAK2 inhibition suppressed MEK/ERK activation in MPN cell lines in vitro, but not in Jak2V617F and MPLW515L mouse models in vivo. JAK2 inhibition ex vivo inhibited MEK/ERK signaling suggesting cell extrinsic factors maintain ERK activation in vivo. We identified PDGFRα as an activated kinase that remains activated upon JAK2 inhibition in vivo, and PDGF-AA/PDGF-BB production persisted in the setting of JAK kinase inhibition. PDGF-BB maintained ERK activation in presence of ruxolitinib consistent with its function as a ligand-induced bypass for ERK activation. Combined JAK/MEK inhibition suppressed MEK/ERK activation in Jak2V617F and MPLW515L mice with increased efficacy and reversal of fibrosis to an extent not seen with JAK inhibitors. This demonstrates that compensatory ERK activation limits the efficacy of JAK2 inhibition and dual JAK/MEK inhibition provides an opportunity for improved therapeutic efficacy in MPNs and in other malignancies driven by aberrant JAK-STAT signaling.
Simona Stivala, Tamara Codilupi, Sime Brkic, Anne Baerenwaldt, Nilabh Ghosh, Hui Hao-Shen, Stephan Dirnhofer, Matthias S. Dettmer, Cedric Simillion, Beat A. Kaufmann, Sophia Chiu, Matthew D. Keller, Maria Kleppe, Morgane Hilpert, Andreas S. Buser, Jakob R. Passweg, Thomas Radimerski, Radek C. Skoda, Ross L. Levine, Sara C. Meyer
Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a three-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding β3 integrin. Crossing msuPAR2-transgenic mice with three different integrin β3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for β3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, mouse uPAR isoform 2 may play an important role in certain forms of scarring kidney disease.
Changli Wei, Jing Li, Brian D. Adair, Ke Zhu, Jian Cai, Michael Merchant, Beata Samelko, Zhongji Liao, Kwi Hye Koh, Nicholas J. Tardi, Ranadheer R. Dande, Shuangxin Liu, Jianchao Ma, Salvatore DiBartolo, Stefan Hägele, Vasil Peev, Salim S. Hayek, David J. Cimbaluk, Melissa Tracy, Jon B. Klein, Sanja Sever, Sanford J. Shattil, M. Amin Arnaout, Jochen Reiser
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