Research
Abstract
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.
Authors
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
Abstract
The discovery of recurrent mutations in subunits of the vacuolar-type H+-translocating ATPase (v-ATPase) in follicular lymphoma (FL) highlights a role for the amino acid- and energy-sensing pathway to MTOR in the pathogenesis of this disease. Here, through the use of complementary experimental approaches involving mammalian cells and Saccharomyces cerevisiae, we have demonstrated that mutations in the v-ATPase subunit ATP6V1B2/Vma2 activate autophagic flux and maintain MTOR/Tor in an active state. Engineered lymphoma cell lines and primary follicular lymphoma B cells (FL B cells) carrying mutated ATP6V1B2 demonstrated a remarkable ability to survive low leucine concentrations. The treatment of primary FL B cells with inhibitors of autophagy uncovered an addiction for survival for FL B cells harboring ATP6V1B2 mutants. These data support mutational activation of autophagic flux by recurrent hotspot mutations in ATP6V1B2 as an adaptive mechanism in FL pathogenesis and as a new possible therapeutically targetable pathway.
Authors
Fangyang Wang, Damián Gatica, Zhang Xiao Ying, Luke F. Peterson, Peter K. Kim, Denzil Bernard, Kamlai Saiya-Cork, Shaomeng Wang, Mark S. Kaminski, Alfred E. Chang, Tycel Phillips, Daniel J. Klionsky, Sami N. Malek
RABL6A inhibits tumor-suppressive PP2A/AKT signaling to drive pancreatic neuroendocrine tumor growth
Abstract
Hyperactivated AKT/mTOR signaling is a hallmark of pancreatic neuroendocrine tumors (PNETs). Drugs targeting this pathway are used clinically but tumor resistance invariably develops. A better understanding of factors regulating AKT/mTOR signaling and PNET pathogenesis is needed to improve current therapies. We discovered that RABL6A, a new oncogenic driver of PNET proliferation, is required for AKT activity. Silencing RABL6A caused PNET cell cycle arrest that coincided with selective loss of AKT-S473 (not T308) phosphorylation and AKT/mTOR inactivation. Restoration of AKT phosphorylation rescued the G1 phase block triggered by RABL6A silencing. Mechanistically, loss of AKT-S473 phosphorylation in RABL6A depleted cells resulted from increased protein phosphatase 2A (PP2A) activity. Inhibition of PP2A restored phosphorylation of AKT-S473 in RABL6A depleted cells whereas PP2A reactivation using a specific small molecule activator of PP2A (SMAP) abolished that phosphorylation. Moreover, SMAP treatment effectively killed PNET cells in a RABL6A-dependent manner and suppressed PNET growth in vivo. This work identifies RABL6A as a new inhibitor of the PP2A tumor suppressor and essential activator of AKT in PNET cells. Our findings offer what we believe is a novel strategy of PP2A reactivation for treatment of PNETs as well as other human cancers driven by RABL6A overexpression and PP2A inactivation.
Authors
Shaikamjad Umesalma, Courtney A. Kaemmer, Jordan L. Kohlmeyer, Blake L. Letney, Angela M. Schab, Jacqueline A. Reilly, Ryan M. Sheehy, Jussara Hagen, Nitija Tiwari, Fenghuang Zhan, Mariah R. Leidinger, Thomas M. O'Dorisio, Joseph S. Dillon, Ronald A. Merrill, David K. Meyerholz, Abbey L. Perl, Bart J. Brown, Terry A. Braun, Aaron T. Scott, Timothy Ginader, Agshin F. Taghiyev, Gideon K. Zamba, James R. Howe, Stefan Strack, Andrew M. Bellizzi, Goutham Narla, Benjamin W. Darbro, Frederick W. Quelle, Dawn E. Quelle
Abstract
Although ccRCC has been shown to have widespread aberrant cytosine methylation and loss of hydroxymethylation (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was significantly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression free survival after resection of non-metastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of TET enzymes, but by their functional inactivation by l-2-hydroxyglutarate (L2HG) that was overexpressed due to the deletion and under-expression of l-2-hydroxyglutarate dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high dose AA in ccRCC.
Authors
Niraj Shenoy, Tushar D. Bhagat, John C. Cheville, Christine Lohse, Sanchari Bhattacharyya, Alexander Tischer, Venkata Machha, Shanisha Gordon-Mitchell, Gaurav S. Choudhary, Li-Fan Wong, LouAnn Gross, Emily Ressegue, Bradley C. Leibovich, Stephen A. Boorjian, Ulrich Steidl, Xiaosheng Wu, Kith Pradhan, Benjamin Gartrell, Beamon Agarwal, Lance Pagliaro, Masako Suzuki, John M. Greally, Dinesh Rakheja, R. Houston Thompson, Katalin Susztak, Thomas Witzig, Yiyu Zou, Amit Verma
Abstract
Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand/receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and anti-tumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells co-engineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior anti-tumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.
Authors
Tori N. Yamamoto, Ping-Hsien Lee, Suman K. Vodnala, Devikala Gurusamy, Rigel J. Kishton, Zhiya Yu, Arash Eidizadeh, Robert Eil, Jessica Fioravanti, Luca Gattinoni, James N. Kochenderfer, Terry J. Fry, Bulent Arman Aksoy, Jeffrey Hammerbacher, Anthony C. Cruz, Richard M. Siegel, Nicholas P. Restifo, Christopher A. Klebanoff
Abstract
Post-stroke cognitive impairment is considered one of the main complications during the chronic phase of ischemic stroke. In the adult brain, the hippocampus regulates both encoding and retrieval of new information through adult neurogenesis. Nevertheless, the lack of predictive models and studies based on the forgetting processes hinder the understanding of memory alterations after stroke. Our aim was to explore whether post-stroke neurogenesis participates in the development of long-term memory impairment. Here we show a hippocampal neurogenesis burst that persisted one month after stroke and that correlated with an impaired contextual and spatial memory performance. Furthermore, we demonstrate that the enhancement of hippocampal neurogenesis after stroke by physical activity or memantine treatment weakened existing memories. More importantly, stroke-induced newborn neurons promoted an aberrant hippocampal circuitry remodelling with differential features at ipsi- and contralesional levels. Strikingly, inhibition of stroke-induced hippocampal neurogenesis by temozolomide treatment or using a genetic approach (Nestin-CreERT2/NSE-DTA mice) impeded the forgetting of old memories. These results suggest that hippocampal neurogenesis modulation could be considered as a potential approach for post-stroke cognitive impairment.
Authors
María Isabel Cuartero, Juan de la Parra, Alberto Pérez-Ruiz, Isabel Bravo-Ferrer, Violeta Durán-Laforet, Alicia García-Culebras, Juan Manuel García-Segura, Jagroop Dhaliwal, Paul W. Frankland, Ignacio Lizasoain, María Àngeles Moro
Abstract
The cytoplasmic aggregation of TDP-43 is a hallmark of degenerating neurons in amyotrophic lateral sclerosis (ALS) and subsets of frontotemporal dementia (FTD). In order to reduce TDP-43 pathology, we have generated single chain (scFv) antibodies against the RNA recognition motif 1 (RRM1) of TDP-43 which is involved in abnormal protein self-aggregation and interaction with p65 nuclear factor kappa B (NFKB). Viral-mediated delivery into the nervous system of a scFv antibody, named VH7Vk9, reduced microgliosis in a mouse model of acute neuroinflammation and it mitigated cognitive impairment, motor defects, TDP-43 proteinopathy and neuroinflammation in transgenic mice expressing ALS-linked TDP-43 mutations. These results suggest that antibodies targeting the RRM1 domain of TDP-43 might provide new therapeutic avenues for treatment of ALS and FTD.
Authors
Silvia Pozzi, Sai Sampath Thammisetty, Philippe Codron, Reza Rahimian, Karine V. Plourde, Geneviève Soucy, Christine Bareil, Daniel Phaneuf, Jasna Kriz, Claude Gravel, Jean-Pierre Julien
Abstract
In the stomach, chronic inflammation causes metaplasia and creates a favorable environment for the evolution of gastric cancer. Glucocorticoids are steroid hormones that repress proinflammatory stimuli but their role in the stomach is unknown. In this study, we show that endogenous glucocorticoids are required to maintain gastric homeostasis. Removal of circulating glucocorticoids in mice by adrenalectomy resulted in the rapid onset of spontaneous gastric inflammation, oxyntic atrophy, and spasmolytic polypeptide-expressing metaplasia (SPEM), a precursor of gastric cancer. SPEM and oxyntic atrophy occurred independently of lymphocytes. However, depletion of monocytes and macrophages by clodronate treatment or inhibition of gastric monocyte infiltration using the Cx3cr1 knockout mouse model prevented SPEM development. Our results highlight the requirement for endogenous glucocorticoid signaling within the stomach to prevent spontaneous gastric inflammation and metaplasia and suggest that glucocorticoid deficiency may lead to gastric cancer development.
Authors
Jonathan T. Busada, Sivapriya Ramamoorthy, Derek W. Cain, Xiaojiang Xu, Donald N. Cook, John A. Cidlowski
Abstract
We used the cancer intrinsic property of oncogene-induced DNA damage as the base for a conditional synthetic lethality approach. To target mechanisms important for cancer cell adaptation to genotoxic stress and thereby to achieve cancer cell-specific killing, we combined inhibition of the kinases ATR and Wee1. Wee1 regulates cell cycle progression, whereas ATR is an apical kinase in the DNA damage response. In an orthotopic breast cancer model, tumor-selective synthetic lethality between bioavailable ATR and Wee1 inhibitors led to tumor remission and inhibited metastasis with minimal side effects. ATR and Wee1 inhibition had a higher synergistic effect in cancer stem cells than in bulk cancer cells, compensating for the lower sensitivity of cancer stem cells to the individual drugs. Mechanistically, the combination treatment caused cells with unrepaired or under-replicated DNA to enter mitosis leading to mitotic catastrophe. As these inhibitors of ATR and Wee1 are already in phase I/II clinical trials, this knowledge could soon be translated into the clinic, especially as we showed that the combination treatment targets a wide range of tumor cells. Particularly the anti-metastatic effect of combined Wee1/ATR inhibition and the low toxicity of ATR inhibitors compared to Chk1 inhibitors has great clinical potential.
Authors
Amirali B. Bukhari, Cody W. Lewis, Joanna J. Pearce, Deandra Luong, Gordon K. Chan, Armin M. Gamper
Abstract
Upon arterial injury, endothelial denudation leads to platelet activation, and delivery of multiple agents (e.g. TXA2, PDGF) promoting VSMC dedifferentiation, and proliferation, in injury repair (intimal hyperplasia). Resolution of vessel injury repair, and prevention of excessive repair (switching VSMC back to a differentiated quiescent state) is a poorly understood process. We now report that internalization of activated platelets by VSMCs promotes resolution of arterial injury by switching on VSMC quiescence. Ex vivo and in vivo studies using lineage tracing reporter mice (PF4-Cre x mTmG) demonstrated uptake of green platelets by red vascular smooth muscle cells upon arterial wire injury. Genome-wide miRNA sequencing of VSMCs co-cultured with activated platelets identified significant increases in platelet-derived miR-223. miR-223 appears to directly target PDGFRβ (in VSMCs) reversing the injury-induced dedifferentiation. Upon arterial injury platelet miR-223 knockout mice exhibit increased intimal hyperplasia, whereas miR-223 mimics reduced intimal hyperplasia. Diabetic mice with reduced expression of miR-223, exhibited enhanced VSMC dedifferentiation, proliferation, and increased intimal hyperplasia. Horizontal transfer of platelet-derived miRNAs into VSMCs provide a novel mechanism for regulating VSMC phenotypic switching. Platelets thus play a dual role in vascular injury repair, initiating an immediate repair process, and concurrently, a delayed process to prevent excessive repair.
Authors
Zhi Zeng, Luoxing Xia, Xuejiao Fan, Allison C. Ostriker, Timur Yarovinsky, Meiling Su, Yuan Zhang, Xiangwen Peng, Xie Yi, Lei Pi, Xiaoqiong Gu, Sookja Kim Chung, Kathleen A. Martin, Renjing Liu, John Hwa, Wai Ho Tang
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ISSN: 0021-9738 (print), 1558-8238 (online)