The tumor microenvironment presents physical, immunologic, and metabolic barriers to durable immunotherapy responses. We have recently described roles for both T cell metabolic insufficiency as well as tumor hypoxia as inhibitory mechanisms which prevent T cell activity in murine tumors, but whether intratumoral T cell activity or response to immunotherapy vary between patients as a function of distinct metabolic profiles in tumor cells remains unclear. Here we show that metabolic derangement can vary widely in both degree and type in patient-derived cell lines and in ex vivo analysis of patient samples, such that some cells demonstrate solely deregulated oxidative or glycolytic metabolism. Further, deregulated oxidative, but not glycolytic, metabolism was associated with increased generation of hypoxia upon implantation into immunodeficient animals. Generation of murine single cell melanoma cell lines that lacked either oxidative or glycolytic metabolism showed that elevated tumor oxygen consumption was associated with increased T cell exhaustion and decreased immune activity. Further, melanoma lines lacking oxidative metabolism were solely responsive to anti-PD1 therapy among those tested. Prospective analysis of patient samples immunotherapy revealed that oxidative, but not glycolytic, metabolism was associated with progression on PD-1 blockade. Our data highlight a role for oxygen as a crucial metabolite required for the tumor-infiltrating T cells to differentiate appropriately upon PD-1 blockade, and suggesting tumor oxidative metabolism may be a target to improve immunotherapeutic response.
Yana G. Najjar, Ashley V. Menk, Cindy Sander, Uma Rao, Arivarasan Karunamurthy, Roma Bhatia, Shuyan Zhai, John M. Kirkwood, Greg M. Delgoffe
Beta-2 microglobulin (β2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but β2M may also have less understood chaperoneindependent functions. Elevated plasma β2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. β2M mRNA is present in platelets at very high levels and β2M is part of the activated platelet releasate. In addition to their more well studied thrombotic functions, platelets are important immune regulatory cells that release inflammatory molecules and contribute to leukocyte trafficking, activation, and differentiation. We have now found that platelet-derived β2M is a mediator of monocyte pro-inflammatory differentiation through non-canonical TGF-β receptor signaling. Circulating monocytes from mice lacking β2M only in platelets (Plt-β2M–/–) had a more pro-reparative monocyte phenotype, in part dependent on increased platelet-derived TGF-β signaling in the absence of β2M. Using a mouse myocardial infarction (MI) model, Plt-β2M–/– mice had limited post-MI pro-inflammatory monocyte responses, and instead demonstrated early pro-reparative monocyte differentiation, profibrotic myofibroblast responses, and a rapid decline in heart function compared to WT mice. These data demonstrate a novel chaperone-independent, monocyte phenotype regulatory function for platelet β2M, and that platelet-derived β2M and TGF-β have opposing roles in monocyte differentiation that may be important in tissue injury responses.
Zachary T. Hilt, Daphne N. Pariser, Sara K. Ture, Amy Mohan, Pearl Quijada, Akua Asante, Scott J. Cameron, Julie A. Sterling, Alyssa R. Merkel, Andrew L. Johanson, Jermaine L. Jenkins, Eric M. Small, Kathleen E. McGrath, James Palis, Michael R. Elliott, Craig N. Morrell
Molecular profiling of prostate cancer with liquid biopsies such as circulating tumor cells (CTC) and cell-free nucleic acid analysis yields informative yet distinct datasets. Additional insights may be gained by simultaneously interrogating multiple liquid biopsy components to construct a more comprehensive molecular disease profile. We conducted an initial proof of principle study aimed at piloting this multi-parametric approach. Peripheral blood samples from men with metastatic castrate resistant prostate cancer (mCRPC) were analyzed simultaneously for CTC enumeration, single cell copy number variation, CTC DNA and matched cell-free DNA mutations, and plasma cell-free RNA levels of androgen receptor (AR) and AR splice variant (AR-V7). In addition, liquid biopsies were compared with matched tumor profiles when available, and a second liquid biopsy was drawn and analyzed at disease progression in a subset of patients. In this manner, multiparametric liquid biopsy profiles were successfully generated for each patient and time point, demonstrating the feasibility of this approach and highlighting shared as well as unique cancer-relevant alterations. With further refinement and validation in large cohorts, multi-parametric liquid biopsies can optimally integrate disparate but clinically informative datasets and maximize their utility for molecularly directed, real-time patient management.
Emmanuelle Hodara, Gareth Morrison, Alexander T. Cunha, Daniel Zainfeld, Tong Xu, Yucheng Xu, Paul W. Dempsey, Paul C. Pagano, Farideh Bischoff, Aditi Khurana, Bonik S. Koo, Marc J. Ting, Philip D. Cotter, Matthew W. Moore, Shelly Gunn, Joshua Usher, Shahrooz Rabizadeh, Peter Danenberg, Kathleen Danenberg, John Carpten, Tanya B. Dorff, David I. Quinn, Amir Goldkorn
Soluble STimulation-2 (ST2) is increased during graft-versus-host disease (GVHD) while regulatory T cells (Tregs) that express ST2 prevent GVHD through unknown mechanisms. Transplantation of Foxp3- T cells and Tregs sorted from different Foxp3 reporter mice indicated that ST2+Tregs isolated from GVHD mice were thymus-derived and predominantly intestine localized. ST2-/- Tregs transplantation was associated with reduced total intestinal Tregs frequency and activation. ST2-/- vs wild-type intestinal Tregs transcriptomes showed decreased Treg functional markers and reciprocally, increased Rorc expression. Rorc-/- T cells transplantation enhanced the frequency and function of intestinal ST2+Tregs and reduced GVHD through decreased gut-infiltrating soluble ST2-producing type-1 and increased IL-4+IL-10+ producing type-2 T cells. Cotransfer of ST2+Tregs sorted from Rorc-/- mice with WT CD25-depleted T cells decreased GVHD severity and mortality, increased intestinal ST2+KLRG1+ Tregs and decreased type-1 T cells after transplantation, indicating an intrinsic mechanism. Ex vivo IL-33 stimulated Tregs (TregIL-33) expressed higher amphiregulin, displayed better immunosuppression, and adoptive transfer prevented GVHD better than control Tregs or TregIL-33 cultured with IL-23/IL-17. Amphiregulin blockade by neutralizing antibody in vivo abolished the protective effect of TregIL-33. Our data show an inversely expression of ST2 and RORγt in intestinal Tregs, and TregIL-33 is a potential cellular therapy avenue for preventing GVHD.
Jinfeng Yang, Abdulraouf Ramadan, Dawn K. Reichenbach, Michael Loschi, Jilu Zhang, Brad Griesenauer, Hong Liu, Keli L. Hippen, Bruce R. Blazar, Sophie Paczesny
Macrophage activation, i.e., the classical M1 and the alternative M2, plays a critical role in many pathophysiological processes, such as inflammation and tissue injury and repair. Although the regulation of macrophage activation has been under extensive investigations, there is little knowledge about the role of long non-coding RNAs (lncRNAs) in the event. In this study, we found that lncRNA Malat1 expression is distinctly regulated in differentially activated macrophages in that it is upregulated in LPS-, whereas downregulated in IL-4-treated cells. Malat1 knockdown attenuates LPS induced M1 macrophage activation. In contrast, Malat1 knockdown enhanced IL-4 activated M2 differentiation as well as macrophage pro-fibrotic phenotype. Mechanistically, Malat1 knockdown led to decreased expression of Clec16a, of which silencing phenocopied the regulatory effect of Malat1 on M1 activation. Interestingly, Malat1 knockdown promoted IL-4 induction of mitochondrial pyruvate carriers (MPCs) and their mediation of glucose derived oxidative phosphorylation (OxPhos), which was crucial to the Malat1 regulation of M2 differentiation and pro-fibrotic phenotype. Furthermore, mice with either global or conditional myeloid knockout of Malat1 demonstrated diminished LPS induced systemic and pulmonary inflammation and injury. Conversely, these mice developed more severe bleomycin induced lung fibrosis, accompanied by alveolar macrophages displaying augmented M2 and pro-fibrotic phenotype. In summary, we have identified a previously unrecognized role of Malat1 in regulation of macrophage polarization. Our data demonstrate that Malat1 is involved in pulmonary pathogeneses in association with aberrant macrophage activation.
Huachun Cui, Sami Banerjee, Sijia Guo, Na Xie, Jing Ge, Dingyuan Jiang, Martin Zörnig, Victor J. Thannickal, Gang Liu
The lymphatic system plays crucial roles in tissue homeostasis, lipid absorption and immune cell trafficking. While lymphatic valves ensure unidirectional lymph flows, the flow itself controls lymphatic valve formation. Here, we demonstrate that a mechanically activated ion channel Piezo1 senses oscillating shear stress (OSS), and incorporates the signal into the genetic program controlling lymphatic valve development and maintenance. Time-controlled deletion of Piezo1 using a pan-endothelial Cre driver (Cdh5(PAC)-CreERT2) or lymphatic-specific Cre driver (Prox1-CreERT2) equally inhibited lymphatic valve formation in newborn mouse. Furthermore, Piezo1 deletion in adult lymphatics caused substantial lymphatic valve degeneration. Piezo1 knockdown in cultured lymphatic endothelial cells (LECs) largely abrogated the OSS-induced upregulation of the lymphatic valve-signature genes. Conversely, ectopic Piezo1 overexpression upregulated the lymphatic valve genes in the absence of OSS. Remarkably, activation of Piezo1 using a chemical agonist Yoda1 not only accelerated lymphatic valve formation in animals, but also triggered upregulation of some lymphatic valve genes in cultured LECs without exposure to OSS. In summary, our studies together demonstrate that Piezo1 is the force sensor in the mechanotransduction pathway controlling lymphatic valve development and maintenance, and Piezo1 activation is a potential novel therapeutic strategy for congenital and surgery-associated lymphedema.
Dongwon Choi, Eunkyung Park, Eunson Jung, Boksik Cha, Somin Lee, James Yu, Paul M. Kim, Sunju Lee, Yeo Jin Hong, Chester J. Koh, Chang-Won Cho, Yifan Wu, Noo Li Jeon, Alex K. Wong, Laura Shin, S. Ram Kumar, Ivan Bermejo-Moreno, R. Sathish Srinivasan, Il-Taeg Cho, Young-Kwon Hong
RNA binding proteins represent an emerging class of proteins with a role in cardiac dysfunction. We show that activation of the RNA binding protein Human antigen R (HuR) is increased in the failing human heart. To determine the functional role of HuR in pathological cardiac hypertrophy, we created an inducible cardiomyocyte-specific HuR deletion mouse, and showed that HuR deletion reduces left ventricular hypertrophy, dilation, and fibrosis while preserving cardiac function in a transverse aortic constriction (TAC) model of pressure-overload-induced hypertrophy. Assessment of HuR-dependent changes in global gene expression suggests that the mechanistic basis for this protection occurs through a reduction in fibrotic signaling, specifically through a reduction in transforming growth factor beta (Tgfb) expression. Finally, pharmacological inhibition of HuR at a clinically relevant time point following the initial development of pathological hypertrophy post-TAC also yielded a significant reduction in pathological progression, as marked by a reduction in hypertrophy, dilation, and fibrosis, and preserved function. In summary, this study demonstrates a functional role for HuR in the progression of pressure overload-induced cardiac hypertrophy and establishes HuR inhibition as a viable therapeutic approach for pathological cardiac hypertrophy and heart failure.
Lisa C. Green, Sarah R. Anthony, Samuel Slone, Lindsey Lanzillotta, Michelle L. Nieman, Xiaoqing Wu, Nathan Robbins, Shannon M. Jones, Sudeshna Roy, A. Phillip Owens III, Jeffrey Aube, Liang Xu, John N. Lorenz, Burns C. Blaxall, Jack Rubinstein, Joshua B. Benoit, Michael Tranter
Diarrhea is a major side effect of ErbB receptor tyrosine kinase inhibitors (TKIs) in cancer chemotherapy. Here, we show that the primary mechanism of ErbB TKI diarrhea is activation of basolateral membrane potassium (K+) channels and apical membrane chloride (Cl-) channels in intestinal epithelia, and demonstrate the efficacy of channel blockers in a rat model of TKI diarrhea. Short-circuit current in colonic epithelial cells showed that the TKIs gefitinib, lapatinib and afatinib do not affect basal secretion, but amplify carbachol-stimulated secretion by 2 to 3 fold. Mechanistic studies with the second-generation TKI afatinib showed that the amplifying effect on Cl- secretion was Ca2+ and cAMP independent, blocked by CFTR and K+ channel inhibitors, and involved the EGF receptor binding and ERK signaling. Afatinib-amplified activation of basolateral K+ and apical Cl- channels was demonstrated by selective membrane permeabilization, ion substitution and channel inhibitors. Rats administered afatinib orally at 60 mg/kg/day developed diarrhea with increased stool water from ~60% to >80%, which was reduced by up to 75% the K+ channel inhibitors clotrimazole or senicapoc, or the CFTR inhibitor (R)-BPO-27. These results indicate a mechanism for TKI diarrhea involving K+ and Cl- channel activation, and support the therapeutic efficacy of channel inhibitors.
Tianying Duan, Onur Cil, Jay R. Thiagarajah, Alan S Verkman
The angiopoietin (Ang)-Tie2 signaling pathway is essential for maintaining vascular homeostasis and its dysregulation is associated with several diseases. Interactions between Tie2 and α5β1 integrin have emerged as part of this control; however, the mechanism is incompletely understood. AXT107, a collagen IV-derived peptide, has strong anti-permeability activity and has enabled the elucidation of this previously undetermined mechanism. Previously, AXT107 was shown to inhibit VEGFR2 and other growth factor signaling via receptor tyrosine kinase association with specific integrins. AXT107 disrupts α5β1 and stimulates the relocation of Tie2 and α5 to cell junctions. In the presence of Ang2 and AXT107, junctional Tie2 is activated, downstream survival signals are upregulated, F-actin is rearranged to strengthen junctions, and, as a result, endothelial junctional permeability is reduced. These data suggest that α5β1 sequesters Tie2 in non-junctional locations in endothelial cell membranes and that AXT107-induced disruption of α5β1 promotes clustering of Tie2 at junctions and converts Ang2 into a strong agonist, similar to responses observed when Ang1 levels greatly exceed those of Ang2. The potentiation of Tie2 activation by Ang2 even extended in to mouse models in which AXT107 induced Tie2 phosphorylation in a model of hypoxia and inhibited vascular leakage in an Ang2-overexpression transgenic model and an LPS-induced inflammation model. Since Ang2 levels are very high in ischemic diseases, such as diabetic macular edema, neovascular age-related macular degeneration, uveitis, and cancer, targeting α5β1 with AXT107 provides a novel and potentially more effective approach to treat these diseases.
Adam C. Mirando, Jikui Shen, Raquel Lima e Silva, Zenny Chu, Nicholas Sass, Valeria E. Lorenc, Jordan J. Green, Peter A. Campochiaro, Aleksander S. Popel, Niranjan B. Pandey
Abnormal activation of neddylation modification and dysregulated energy metabolism are frequently seen in many types of cancer cells. Whether and how neddylation modification affects cellular metabolism remains largely unknown. Here we showed that MLN4924, a small molecule inhibitor of neddylation modification, induces mitochondrial fission-to-fusion conversion in breast cancer cells via inhibiting ubiquitylation and degradation of fusion-promoting protein mitofusin (MFN1) by SCFβ-TrCP E3 ligase and blocking the mitochondrial translocation of fusion-inhibiting protein DRP1. Importantly, MLN4924-induced mitochondrial fusion is independent of cell cycle progression, but confers cellular survival. The Mass-Spectrometry-based metabolic profiling and mitochondrial functional assays reveal that MLN4924 inhibits TCA cycle, but promotes mitochondrial OXPHOS. MLN4924 also increases glycolysis by activating PKM2 via promoting its tetramerization. Biologically, MLN4924 coupled with OXPHOS inhibitor metformin, or glycolysis inhibitor shikonin, significantly inhibits cancer cell growth both in vitro and in vivo. Together, our study links neddylation modification and energy metabolism, and provides sound strategies for effective combinational cancer therapies.
Qiyin Zhou, Hua Li, Yuanyuan Li, Mingjia Tan, Shaohua Fan, Cong Cao, Feilong Meng, Ling Zhu, Lili Zhao, Min-Xin Guan, Hongchuan Jin, Yi Sun
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