In this issue of JCI Insight, Korchynska et al. demonstrate that the lipid-sensing orphan receptor GPR55 localizes to self-renewing ductal epithelial cells and their terminally-differentiated progeny in salivary glands and is a key regulator of salivation. The cover image is a confocal laser-scanning micrograph of granulated ducts from a murine submandibular salivary gland, showing GPR55 (green), protein disulfide isomerase (red), and lectin (in blue). Hoechst 33,342, was used as nuclear counterstain (white)
Small heat shock proteins (sHSPs) comprise an important protein family that is ubiquitously expressed, is highly conserved among species, and has emerged as a critical regulator of protein folding. While these proteins are functionally important for a variety of tissues, an emerging field of cardiovascular research reveals sHSPs are also extremely important for maintaining normal cardiac function and regulating the cardiac stress response. Notably, numerous mutations in genes encoding sHSPs have been associated with multiple cardiac diseases. sHSPs (HSPB5, HSPB6, and HSPB8) have been described as mediating chaperone functions within the heart by interacting with the cochaperone protein BCL-2–associated anthanogene 3 (BAG3); however, recent reports indicate that sHSPs (HSPB7) can perform other BAG3-independent functions. Here, we summarize the cardiac functions of sHSPs and present the notion that cardiac sHSPs function via BAG3-dependent or -independent pathways.
Xi Fang, Julius Bogomolovas, Christa Trexler, Ju Chen
Immunotherapy has emerged as a promising approach to treat cancer. However, partial responses across multiple clinical trials support the significance of characterizing intertumor and intratumor heterogeneity to achieve better clinical results and as potential tools in selecting patients for different types of cancer immunotherapies. Yet, the type of heterogeneity that informs clinical outcome and patient selection has not been fully explored. In particular, the lack of characterization of immune response–related genes in cancer cells hinders the further development of metrics to select and optimize immunotherapy. Therefore, we analyzed single-cell RNA-Seq data from lung adenocarcinoma patients and cell lines to characterize the intratumor heterogeneity of immune response–related genes and demonstrated their potential impact on the efficacy of immunotherapy. We discovered that IFN-γ signaling pathway genes are heterogeneously expressed and coregulated with other genes in single cancer cells, including MHC class II (MHCII) genes. The downregulation of genes in IFN-γ signaling pathways in cell lines corresponds to an acquired resistance phenotype. Moreover, analysis of 2 groups of tumor-restricted antigens, namely neoantigens and cancer testis antigens, revealed heterogeneity in their expression in single cells. These analyses provide a rationale for applying multiantigen combinatorial therapies to prevent tumor escape and establish a basis for future development of prognostic metrics based on intratumor heterogeneity.
Ke-Yue Ma, Alexandra A. Schonnesen, Amy Brock, Carla Van Den Berg, S. Gail Eckhardt, Zhihua Liu, Ning Jiang
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 TGF-β (Tgfb) expression. Finally, pharmacological inhibition of HuR at a clinically relevant time point following the initial development of pathological hypertrophy after 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
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 1 (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. Mass-spectrometry-based metabolic profiling and mitochondrial functional assays reveal that MLN4924 inhibits the TCA cycle but promotes mitochondrial OXPHOS. MLN4924 also increases glycolysis by activating PKM2 via promoting its tetramerization. Biologically, MLN4924 coupled with the OXPHOS inhibitor metformin, or the 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 combined 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
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 antipermeability 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 nonjunctional 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 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. Because 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 potentially more effective approach to treat these diseases.
Adam C. Mirando, Jikui Shen, Raquel Lima e Silva, Zenny Chu, Nicholas C. Sass, Valeria E. Lorenc, Jordan J. Green, Peter A. Campochiaro, Aleksander S. Popel, Niranjan B. Pandey
Acute cardiorenal syndrome (CRS-1) is a morbid complication of acute cardiovascular disease. Heart-to-kidney signals transmitted by “cardiorenal connectors” have been postulated, but investigation into CRS-1 has been limited by technical limitations and a paucity of models. To address these limitations, we developed a translational model of CRS-1, cardiac arrest and cardiopulmonary resuscitation (CA/CPR), and now report findings from nanoscale mass spectrometry proteomic exploration of glomerular filtrate 2 hours after CA/CPR or sham procedure. Filtrate acquisition was confirmed by imaging, molecular weight and charge distribution, and exclusion of protein specific to surrounding cells. Filtration of proteins specific to the heart was detected following CA/CPR and confirmed with mass spectrometry performed using urine collections from mice with deficient tubular endocytosis. Cardiac LIM protein was a CA/CPR-specific filtrate component. Cardiac arrest induced plasma release of cardiac LIM protein in mice and critically ill human cardiac arrest survivors, and administration of recombinant cardiac LIM protein to mice altered renal function. These findings demonstrate that glomerular filtrate is accessible to nanoscale proteomics and elucidate the population of proteins filtered 2 hours after CA/CPR. The identification of cardiac-specific proteins in renal filtrate suggests a novel signaling mechanism in CRS-1. We expect these findings to advance understanding of CRS-1.
Rumie Wakasaki, Katsuyuki Matsushita, Kirsti Golgotiu, Sharon Anderson, Mahaba B. Eiwaz, Daniel J. Orton, Sang Jun Han, H. Thomas Lee, Richard D. Smith, Karin D. Rodland, Paul D. Piehowski, Michael P. Hutchens
GPR55, a lipid-sensing receptor, is implicated in cell cycle control, malignant cell mobilization, and tissue invasion in cancer. However, a physiological role for GPR55 is virtually unknown for any tissue type. Here, we localize GPR55 to self-renewing ductal epithelial cells and their terminally differentiated progeny in both human and mouse salivary glands. Moreover, we find GPR55 expression downregulated in salivary gland mucoepidermoid carcinomas and GPR55 reinstatement by antitumor irradiation, suggesting that GPR55 controls renegade proliferation. Indeed, GPR55 antagonism increases cell proliferation and function determination in quasiphysiological systems. In addition, Gpr55–/– mice present ~50% enlarged submandibular glands with many more granulated ducts, as well as disordered endoplasmic reticuli and with glycoprotein content. Next, we hypothesized that GPR55 could also modulate salivation and glycoprotein content by entraining differentiated excretory progeny. Accordingly, GPR55 activation facilitated glycoprotein release by itself, inducing low-amplitude Ca2+ oscillations, as well as enhancing acetylcholine-induced Ca2+ responses. Topical application of GPR55 agonists, which are ineffective in Gpr55–/– mice, into adult rodent submandibular glands increased salivation and saliva glycoprotein content. Overall, we propose that GPR55 signaling in epithelial cells ensures both the life-long renewal of ductal cells and the continuous availability of saliva and glycoproteins for oral health and food intake.
Solomiia Korchynska, Mirjam I. Lutz, Erzsébet Borók, Johannes Pammer, Valentina Cinquina, Nataliya Fedirko, Andrew J. Irving, Ken Mackie, Tibor Harkany, Erik Keimpema
Newly emerging viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle Eastern respiratory syndrome CoVs (MERS-CoV), and H7N9, cause fatal acute lung injury (ALI) by driving hypercytokinemia and aggressive inflammation through mechanisms that remain elusive. In SARS-CoV/macaque models, we determined that anti–spike IgG (S-IgG), in productively infected lungs, causes severe ALI by skewing inflammation-resolving response. Alveolar macrophages underwent functional polarization in acutely infected macaques, demonstrating simultaneously both proinflammatory and wound-healing characteristics. The presence of S-IgG prior to viral clearance, however, abrogated wound-healing responses and promoted MCP1 and IL-8 production and proinflammatory monocyte/macrophage recruitment and accumulation. Critically, patients who eventually died of SARS (hereafter referred to as deceased patients) displayed similarly accumulated pulmonary proinflammatory, absence of wound-healing macrophages, and faster neutralizing antibody responses. Their sera enhanced SARS-CoV–induced MCP1 and IL-8 production by human monocyte–derived wound-healing macrophages, whereas blockade of FcγR reduced such effects. Our findings reveal a mechanism responsible for virus-mediated ALI, define a pathological consequence of viral specific antibody response, and provide a potential target for treatment of SARS-CoV or other virus-mediated lung injury.
Li Liu, Qiang Wei, Qingqing Lin, Jun Fang, Haibo Wang, Hauyee Kwok, Hangying Tang, Kenji Nishiura, Jie Peng, Zhiwu Tan, Tongjin Wu, Ka-Wai Cheung, Kwok-Hung Chan, Xavier Alvarez, Chuan Qin, Andrew Lackner, Stanley Perlman, Kwok-Yung Yuen, Zhiwei Chen
The phosphaturic hormone FGF23 is elevated in chronic kidney disease (CKD). The risk of premature death is substantially higher in the CKD patient population, with cardiovascular disease (CVD) as the leading mortality cause at all stages of CKD. Elevated FGF23 in CKD has been associated with increased odds for all-cause mortality; however, whether FGF23 is associated with positive adaptation in CKD is unknown. To test the role of FGF23 in CKD phenotypes, a late osteoblast/osteocyte conditional flox-Fgf23 mouse (Fgf23fl/fl/Dmp1-Cre+/–) was placed on an adenine-containing diet to induce CKD. Serum analysis showed casein-fed Cre+ mice had significantly higher serum phosphate and blood urea nitrogen (BUN) versus casein diet and Cre– genotype controls. Adenine significantly induced serum intact FGF23 in the Cre– mice over casein-fed mice, whereas Cre+ mice on adenine had 90% reduction in serum intact FGF23 and C-terminal FGF23 as well as bone Fgf23 mRNA. Parathyroid hormone was significantly elevated in mice fed adenine diet regardless of genotype, which significantly enhanced midshaft cortical porosity. Echocardiographs of the adenine-fed Cre+ hearts revealed profound aortic calcification and cardiac hypertrophy versus diet and genotype controls. Thus, these studies demonstrate that increased bone FGF23, although associated with poor outcomes in CKD, is necessary to protect against the cardio-renal consequences of elevated tissue phosphate.
Erica L. Clinkenbeard, Megan L. Noonan, Joseph C. Thomas, Pu Ni, Julia M. Hum, Mohammad Aref, Elizabeth A. Swallow, Sharon M. Moe, Matthew R. Allen, Kenneth E. White
Evidence has emerged that the failing heart increases utilization of ketone bodies. We sought to determine whether this fuel shift is adaptive. Mice rendered incapable of oxidizing the ketone body 3-hydroxybutyrate (3OHB) in the heart exhibited worsened heart failure in response to fasting or a pressure overload/ischemic insult compared with WT controls. Increased delivery of 3OHB ameliorated pathologic cardiac remodeling and dysfunction in mice and in a canine pacing model of progressive heart failure. 3OHB was shown to enhance bioenergetic thermodynamics of isolated mitochondria in the context of limiting levels of fatty acids. These results indicate that the heart utilizes 3OHB as a metabolic stress defense and suggest that strategies aimed at increasing ketone delivery to the heart could prove useful in the treatment of heart failure.
Julie L. Horton, Michael T. Davidson, Clara Kurishima, Rick B. Vega, Jeffery C. Powers, Timothy R. Matsuura, Christopher Petucci, E. Douglas Lewandowski, Peter A. Crawford, Deborah M. Muoio, Fabio A. Recchia, Daniel P. Kelly
Poly(ADP-ribosyl)ation refers to the covalent attachment of ADP-ribose to protein, generating branched, long chains of ADP-ribose moieties, known as poly(ADP-ribose) (PAR). Poly(ADP-ribose) polymerase 1 (PARP1) is the main polymerase and acceptor of PAR in response to DNA damage. Excessive intracellular PAR accumulation due to PARP1 activation leads cell death in a pathway known as parthanatos. PAR degradation is mainly controlled by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose-acceptor hydrolase 3 (ARH3). Our previous results demonstrated that ARH3 confers protection against hydrogen peroxide (H2O2) exposure, by lowering cytosolic and nuclear PAR levels and preventing apoptosis-inducing factor (AIF) nuclear translocation. We identified a family with an ARH3 gene mutation that resulted in a truncated, inactive protein. The 8-year-old proband exhibited a progressive neurodegeneration phenotype. In addition, parthanatos was observed in neurons of the patient’s deceased sibling, and an older sibling exhibited a mild behavioral phenotype. Consistent with the previous findings, the patient’s fibroblasts and ARH3-deficient mice were more sensitive, respectively, to H2O2 stress and cerebral ischemia/reperfusion-induced PAR accumulation and cell death. Further, PARP1 inhibition alleviated cell death and injury resulting from oxidative stress and ischemia/reperfusion. PARP1 inhibitors may attenuate the progression of neurodegeneration in affected patients with ARH3 deficiency.
Masato Mashimo, Xiangning Bu, Kazumasa Aoyama, Jiro Kato, Hiroko Ishiwata-Endo, Linda A. Stevens, Atsushi Kasamatsu, Lynne A. Wolfe, Camilo Toro, David Adams, Thomas Markello, William A. Gahl, Joel Moss
Macrophage activation, i.e., 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 investigation, there is little knowledge about the role of long noncoding RNAs (lncRNAs) in this event. In this study, we found that lncRNA Malat1 expression is distinctly regulated in differentially activated macrophages in that it is upregulated in LPS-treated and 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 a macrophage profibrotic phenotype. Mechanistically, Malat1 knockdown led to decreased expression of Clec16a, silencing of which 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 profibrotic phenotype. Furthermore, mice with either global or conditional myeloid knockout of Malat1 demonstrated diminished LPS-induced systemic and pulmonary inflammation and injury. In contrast, these mice developed more severe bleomycin-induced lung fibrosis, accompanied by alveolar macrophages displaying augmented M2 and profibrotic phenotypes. In summary, we have identified what we believe is a previously unrecognized role of Malat1 in the 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
Atherosclerosis is a leading cause of death worldwide in industrialized countries. Disease progression and regression are associated with different activation states of macrophages derived from inflammatory monocytes entering the plaques. The features of monocyte-to-macrophage transition and the full spectrum of macrophage activation states during either plaque progression or regression, however, are incompletely established. Here, we use a combination of single-cell RNA sequencing and genetic fate mapping to profile, for the first time to our knowledge, plaque cells derived from CX3CR1+ precursors in mice during both progression and regression of atherosclerosis. The analyses revealed a spectrum of macrophage activation states with greater complexity than the traditional M1 and M2 polarization states, with progression associated with differentiation of CXC3R1+ monocytes into more distinct states than during regression. We also identified an unexpected cluster of proliferating monocytes with a stem cell–like signature, suggesting that monocytes may persist in a proliferating self-renewal state in inflamed tissue, rather than differentiating immediately into macrophages after entering the tissue.
Jian-Da Lin, Hitoo Nishi, Jordan Poles, Xiang Niu, Caroline Mccauley, Karishma Rahman, Emily J. Brown, Stephen T. Yeung, Nikollaq Vozhilla, Ada Weinstock, Stephen A. Ramsey, Edward A. Fisher, P’ng Loke
Allergic eosinophilic asthma is a chronic condition causing airway remodeling resulting in lung dysfunction. We observed that expression of sirtuin 2 (Sirt2), a histone deacetylase, regulates the recruitment of eosinophils after sensitization and challenge with a triple antigen: dust mite, ragweed, and Aspergillus fumigatus (DRA). Our data demonstrate that IL-4 regulates the expression of Sirt2 isoform 3/5. Pharmacological inhibition of Sirt2 by AGK2 resulted in diminished cellular recruitment, decreased CCL17/TARC, and reduced goblet cell hyperplasia. YM1 and Fizz1 expression was reduced in AGK2-treated, IL-4–stimulated lung macrophages in vitro as well as in lung macrophages from AGK2-DRA–challenged mice. Conversely, overexpression of Sirt2 resulted in increased cellular recruitment, CCL17 production, and goblet cell hyperplasia following DRA challenge. Sirt2 isoform 3/5 was upregulated in primary human alveolar macrophages following IL-4 and AGK2 treatment, which resulted in reduced CCL17 and markers of alternative activation. These gain-of-function and loss-of-function studies indicate that Sirt2 could be developed as a treatment for eosinophilic asthma.
Yong Gyu Lee, Brenda F. Reader, Derrick Herman, Adam Streicher, Joshua A. Englert, Mathias Ziegler, Sangwoon Chung, Manjula Karpurapu, Gye Young Park, John W. Christman, Megan N. Ballinger
Diabetes mellitus is associated with various disorders of the locomotor system including the decline in mass and function of skeletal muscle. The mechanism underlying this association has remained ambiguous, however. We now show that the abundance of the transcription factor KLF15 as well as the expression of genes related to muscle atrophy are increased in skeletal muscle of diabetic model mice, and that mice with muscle-specific KLF15 deficiency are protected from the diabetes-induced decline of skeletal muscle mass. Hyperglycemia was found to upregulate the KLF15 protein in skeletal muscle of diabetic animals, which is achieved via downregulation of the E3 ubiquitin ligase WWP1 and consequent suppression of the ubiquitin-dependent degradation of KLF15. Our results revealed that hyperglycemia, a central disorder in diabetes, promotes muscle atrophy via a WWP1/KLF15 pathway. This pathway may serve as a therapeutic target for decline in skeletal muscle mass accompanied by diabetes mellitus.
Yu Hirata, Kazuhiro Nomura, Yoko Senga, Yuko Okada, Kenta Kobayashi, Shiki Okamoto, Yasuhiko Minokoshi, Michihiro Imamura, Shin’ichi Takeda, Tetsuya Hosooka, Wataru Ogawa
Dormant cancer cells known as disseminated tumor cells (DTCs) are often present in bone marrow of breast cancer patients. These DTCs are thought to be responsible for the incurable recurrence of breast cancer. The mechanism underlying the long-term maintenance of DTCs remains unclear, however. Here, we show that Fbxw7 is essential for the maintenance of breast cancer dormancy. Genetic ablation of Fbxw7 in breast cancer cells disrupted the quiescence of DTCs, rendering them proliferative, in mouse xenograft and allograft models. Fbxw7-deficient DTCs were significantly depleted by treatment with paclitaxel, suggesting that cell proliferation induced by Fbxw7 ablation sensitized DTCs to chemotherapy. The combination of Fbxw7 ablation and chemotherapy reduced the number of DTCs even when applied after tumor cell dissemination. Mice injected with Fbxw7-deficient cancer cells survived longer after tumor resection and subsequent chemotherapy than did those injected with wild-type cells. Furthermore, database analysis revealed that breast cancer patients whose tumors expressed FBXW7 at a high level had a poorer prognosis than did those with a low FBXW7 expression level. Our results suggest that a wake-up strategy for DTCs based on Fbxw7 inhibition might be of value in combination with conventional chemotherapy for the treatment of breast cancer.
Hideyuki Shimizu, Shoichiro Takeishi, Hirokazu Nakatsumi, Keiichi I. Nakayama
The rate of decline in insulin secretion after diagnosis with type 1 diabetes (T1D) varies substantially among individuals and with age at diagnosis, but the mechanism(s) behind this heterogeneity are not well understood. We investigated the loss of pancreatic β cell function in new-onset T1D subjects using unbiased whole blood RNA-seq and verified key findings by targeted cell count measurements. We found that patients who lost insulin secretion more rapidly had immune phenotypes (“immunotypes”) characterized by higher levels of B cells and lower levels of neutrophils, especially neutrophils expressing primary granule genes. The B cell and neutrophil immunotypes showed strong age dependence, with B cell levels in particular predicting rate of progression in young subjects only. This age relationship suggested that therapy targeting B cells in T1D would be most effective in young subjects with high pretreatment B cell levels, a prediction which was supported by data from a clinical trial of rituximab in new-onset subjects. These findings demonstrate a link between age-related immunotypes and disease outcome in new-onset T1D. Furthermore, our data suggest that greater success could be achieved by targeted use of immunomodulatory therapy in specific T1D populations defined by age and immune characteristics.
Matthew J. Dufort, Carla J. Greenbaum, Cate Speake, Peter S. Linsley
Although tyrosine kinase inhibitors (TKIs) have demonstrated significant efficacy in advanced lung adenocarcinoma (LUAD) patients with pathogenic alterations in EGFR, most patients develop acquired resistance to these agents via mechanisms enabling the sustained activation of the PI3K and MAPK oncogenic pathways downstream of EGFR. The tumor suppressor protein phosphatase 2A (PP2A) acts as a negative regulator of these pathways. We hypothesize that activation of PP2A simultaneously inhibits the PI3K and MAPK pathways and represents a promising therapeutic strategy for the treatment of TKI-resistant LUAD. After establishing the efficacy of small molecule activators of PP2A (SMAPs) in a transgenic EGFRL858R model and TKI-sensitive cell lines, we evaluated their therapeutic potential in vitro and in vivo in TKI-resistant models. PP2A activation resulted in apoptosis, significant tumor growth inhibition, and downregulation of PI3K and MAPK pathways. Combination of SMAPs and TKI afatinib resulted in an enhanced effect on the downregulation of the PI3K pathway via degradation of the PP2A endogenous inhibitor CIP2A. An improved effect on tumor growth inhibition was observed in a TKI-resistant xenograft mouse model treated with a combination of both agents. These collective data support the development of PP2A activators for the treatment of TKI-resistant LUAD.
Rita Tohmé, Sudeh Izadmehr, Sai Gandhe, Giancarlo Tabaro, Sanjay Vallabhaneni, Ava Thomas, Neal Vasireddi, Neil S. Dhawan, Avi Ma’ayan, Neelesh Sharma, Matthew D. Galsky, Michael Ohlmeyer, Jaya Sangodkar, Goutham Narla
BACKGROUND. Protein disulfide isomerase (PDI) is a thiol isomerase secreted by vascular cells that is required for thrombus formation. Quercetin flavonoids inhibit PDI activity and block platelet accumulation and fibrin generation at the site of a vascular injury in mouse models, but the clinical effect of targeting extracellular PDI in humans has not been studied. METHODS. We conducted a multicenter phase II trial of sequential dosing cohorts to evaluate the efficacy of targeting PDI with isoquercetin to reduce hypercoagulability in cancer patients at high risk for thrombosis. Patients received isoquercetin at 500 mg (cohort A, n = 28) or 1000 mg (cohort B, n = 29) daily for 56 days, with laboratory assays performed at baseline and the end of the study, along with bilateral lower extremity compression ultrasound. The primary efficacy endpoint was a reduction in D-dimer, and the primary clinical endpoint included pulmonary embolism or proximal deep vein thrombosis. RESULTS. The administration of 1000 mg isoquercetin decreased D-dimer plasma concentrations by a median of –21.9% (P = 0.0002). There were no primary VTE events or major hemorrhages observed in either cohort. Isoquercetin increased PDI inhibitory activity in plasma (37.0% in cohort A, n = 25, P < 0.001; 73.3% in cohort B, n = 22, P < 0.001, respectively). Corroborating the antithrombotic efficacy, we also observed a significant decrease in platelet-dependent thrombin generation (cohort A median decrease –31.1%, P = 0.007; cohort B median decrease –57.2%, P = 0.004) and circulating soluble P selectin at the 1000 mg isoquercetin dose (median decrease –57.9%, P < 0.0001). CONCLUSIONS. Isoquercetin targets extracellular PDI and improves markers of coagulation in advanced cancer patients. TRIAL REGISTRATION. Clinicaltrials.gov NCT02195232. FUNDING. Quercegen Pharmaceuticals; National Heart, Lung, and Blood Institute (NHLBI; U54HL112302, R35HL135775, and T32HL007917); and NHLBI Consortium Linking Oncology and Thrombosis (U01HL143365).
Jeffrey I. Zwicker, Benjamin L. Schlechter, Jack D. Stopa, Howard A. Liebman, Anita Aggarwal, Maneka Puligandla, Thomas Caughey, Kenneth A. Bauer, Nancy Kuemmerle, Ellice Wong, Ted Wun, Marilyn McLaughlin, Manuel Hidalgo, Donna Neuberg, Bruce Furie, Robert Flaumenhaft, on behalf of CATIQ Investigators
BACKGROUND. Simultaneous noninvasively recorded skin sympathetic nerve activity (SKNA) and electrocardiogram (neuECG) can be used to estimate cardiac sympathetic tone. We tested the hypothesis that large and prolonged SKNA bursts are associated with temporal clustering arrhythmias. METHODS. We recorded neuECG in 10 patients (69 ± 10 years old) with atrial fibrillation (AF) episodes and in 6 patients (50 ± 13 years old) with ventricular tachycardia (VT) or fibrillation (VF) episodes. Clustering was defined by an arrhythmic episode followed within 1 minute by spontaneous recurrences of the same arrhythmia. The neuECG signals were bandpass filtered between 500–1000 Hz to display SKNA. RESULTS. There were 22 AF clusters, including 231 AF episodes from 6 patients, and 9 VT/VF clusters, including 99 VT/VF episodes from 3 patients. A total duration of SKNA bursts associated with AF was longer than that during sinus rhythm (78.9 min/hour [interquartile range (IQR) 17.5–201.3] vs. 16.3 min/hour [IQR 14.5–18.5], P = 0.022). The burst amplitude associated with AF in clustering patients was significantly higher than that in nonclustering patients (1.54 μV [IQR 1.35–1.89], n = 114, vs. 1.20 μV [IQR 1.05–1.42], n = 21, P < 0.001). The SKNA bursts associated with VT/VF clusters lasted 9.3 ± 3.1 minutes, with peaks that averaged 1.13 ± 0.38 μV as compared with 0.79 ± 0.11 μV at baseline (P = 0.041). CONCLUSION. Large and sustained sympathetic nerve activities are associated with the temporal clustering of AF and VT/VF. FUNDING. This study was supported in part by NIH grants R42DA043391 (THE), R56 HL71140, TR002208-01, R01 HL139829 (PSC), a Charles Fisch Cardiovascular Research Award endowed by Suzanne B. Knoebel of the Krannert Institute of Cardiology (TK and THE), a Medtronic-Zipes Endowment, and the Indiana University Health-Indiana University School of Medicine Strategic Research Initiative (PSC).
Takashi Kusayama, Juyi Wan, Anisiia Doytchinova, Johnson Wong, Ryan A. Kabir, Gloria Mitscher, Susan Straka, Changyu Shen, Thomas H. Everett IV, Peng-Sheng Chen
Antibodies and cytotoxic T cells represent 2 arms of host defense against pathogens. We hypothesized that vaccines that induce both high-magnitude CD8+ T cell responses and antibody responses might confer enhanced protection against HIV. To test this hypothesis, we immunized 3 groups of nonhuman primates: (a) Group 1, which includes sequential immunization regimen involving heterologous viral vectors (HVVs) comprising vesicular stomatitis virus, vaccinia virus, and adenovirus serotype 5–expressing SIVmac239 Gag; (b) Group 2, which includes immunization with a clade C HIV-1 envelope (Env) gp140 protein adjuvanted with nanoparticles containing a TLR7/8 agonist (3M-052); and (c) Group 3, which includes a combination of both regimens. Immunization with HVVs induced very high–magnitude Gag-specific CD8+ T cell responses in blood and tissue-resident CD8+ memory T cells in vaginal mucosa. Immunization with 3M-052 adjuvanted Env protein induced robust and persistent antibody responses and long-lasting innate responses. Despite similar antibody titers in Groups 2 and 3, there was enhanced protection in the younger animals in Group 3, against intravaginal infection with a heterologous SHIV strain. This protection correlated with the magnitude of the serum and vaginal Env-specific antibody titers on the day of challenge. Thus, vaccination strategies that induce both CD8+ T cell and antibody responses can confer enhanced protection against infection.
Caroline Petitdemange, Sudhir Pai Kasturi, Pamela A. Kozlowski, Rafiq Nabi, Clare F. Quarnstrom, Pradeep Babu Jagadeesh Reddy, Cynthia A. Derdeyn, Lori M. Spicer, Parin Patel, Traci Legere, Yevgeniy O. Kovalenkov, Celia C. Labranche, François Villinger, Mark Tomai, John Vasilakos, Barton Haynes, C. Yong Kang, James S. Gibbs, Jonathan W. Yewdell, Dan Barouch, Jens Wrammert, David Montefiori, Eric Hunter, Rama R. Amara, David Masopust, Bali Pulendran
Costimulatory interactions control T cell activation at sites of activated antigen-presenting cells, including B cells. Blockade of the CD28/CD80/CD86 costimulatory axis with CTLA4Ig (abatacept) is widely used to treat certain autoimmune diseases. While transiently effective in subjects with new-onset type 1 diabetes (T1D), abatacept did not induce long-lasting immune tolerance. To elucidate mechanisms limiting immune tolerance in T1D, we performed unbiased analysis of whole blood transcriptomes and targeted measurements of cell subset levels in subjects from a clinical trial of abatacept in new-onset T1D. We showed that individual subjects displayed age-related immune phenotypes (“immunotypes”) at baseline, characterized by elevated levels of B cells or neutrophils, that accompanied rapid or slow progression, respectively, in both abatacept- and placebo-treated groups. A more pronounced immunotype was exhibited by a subset of subjects showing poor response (resistance) to abatacept. This resistance immunotype was characterized by a transient increase in activated B cells (one of the cell types that binds abatacept), reprogrammed costimulatory ligand gene expression, and reduced inhibition of anti-insulin antibodies. Our findings identify immunotypes in T1D subjects that are linked to the rate of disease progression, both in placebo- and abatacept-treated subjects. Furthermore, our results suggest therapeutic approaches to restore immune tolerance in T1D.
Peter S. Linsley, Carla J. Greenbaum, Cate Speake, S. Alice Long, Matthew J. Dufort
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, was blocked by CF transmembrane conductance regulator (CFTR) and K+ channel inhibitors, and involved EGFR 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 that were administered afatinib orally at 60 mg/kg/day developed diarrhea with increased stool water from approximately 60% to greater than 80%, which was reduced by up to 75% by 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
Current clinical methods for the evaluation of lymphatic vessel function, crucial for early diagnosis and evaluation of treatment response of several pathological conditions, in particular of postsurgical lymphedema, are based on complex and mainly qualitative imaging techniques. To address this unmet medical need, we established a simple strategy for the painless and quantitative assessment of cutaneous lymphatic function. We prepared a lymphatic-specific tracer formulation, consisting of the clinically approved near-infrared fluorescent dye, indocyanine green, and the solubilizing surfactant Kolliphor HS15. The tracer was noninvasively delivered to the dermal layer of the skin using MicronJet600 hollow microneedles, and the fluorescence signal decay at the injection site was measured over time using a custom-made, portable detection device. The decay rate of fluorescence signal in the skin was used as a direct measure of lymphatic vessel drainage function. With this method, we could quantify impaired lymphatic clearance in transgenic mice lacking dermal lymphatics and distinguish distinct lymphatic clearance patterns in pigs in different body locations and under manual stimulus. Overall, this method has the potential for becoming a noninvasive and quantitative clinical “office test” for lymphatic function assessment.
Anna K. Polomska, Steven T. Proulx, Davide Brambilla, Daniel Fehr, Mathias Bonmarin, Simon Brändli, Mirko Meboldt, Christian Steuer, Tsvetina Vasileva, Nils Reinke, Jean-Christophe Leroux, Michael Detmar
Alfred L. Garfall, Edward A. Stadtmauer, Wei-Ting Hwang, Simon F. Lacey, Jan Joseph Melenhorst, Maria Krevvata, Martin P. Carroll, William H. Matsui, Qiuju Wang, Madhav V. Dhodapkar, Kavita Dhodapkar, Rituparna Das, Dan T. Vogl, Brendan M. Weiss, Adam D. Cohen, Patricia A. Mangan, Emily C. Ayers, Selene Nunez-Cruz, Irina Kulikovskaya, Megan M. Davis, Anne Lamontagne, Karen Dengel, Naseem D.S. Kerr, Regina M. Young, Donald L. Siegel, Bruce L. Levine, Michael C. Milone, Marcela V. Maus, Carl H. June