The Epstein Barr virus (EBV) is one of the predominant tumor viruses in humans, but so far no therapeutic or prophylactic vaccination against this transforming pathogen is available. We demonstrated that heterologous prime-boost vaccination with the nuclear antigen 1 of EBV (EBNA1) either targeted to the DEC205 receptor on dendritic cells or expressed from a recombinant modified vaccinia virus Ankara (MVA) vector improved priming of antigen-specific CD4+ T-cell help. This help supported the expansion and maintenance of EBNA1 specific CD8+ T cells that are most efficiently primed by recombinant adenoviruses that encode EBNA1. These combined CD4+ and CD8+ T-cell responses protected from EBNA1 expressing T and B cell lymphomas, including lymphoproliferations that emerge spontaneously after EBNA1 expression. In particular the heterologous EBNA1-expressing adenovirus, boosted by EBNA1-encoding MVA vaccination, demonstrated protection as prophylactic and therapeutic treatment of the respective lymphoma challenges. Therefore, we suggest that such heterologous prime-boost vaccinations should be further explored for clinical development against EBV-associated malignancies as well as symptomatic primary EBV infection.
Julia Rühl, Carmen Citterio, Christine Engelmann, Tracey A. Haigh, Andrzej Dzionek, Johannes H. Dreyer, Rajiv Khanna, Graham S. Taylor, Joanna B. Wilson, Carol S. Leung, Christian Münz
A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA-E in tumor cells, which suppresses natural killer (NK) cell activity via ligation of the NK inhibitory receptor CD94/NKG2A. Gene expression data from approximately 10,000 tumor samples showed widespread HLAE expression, with levels correlating with those of KLRC1 (NKG2A) and KLRD1 (CD94). To bypass HLA-E inhibition, we developed a way to generate highly functional NK cells lacking NKG2A. Constructs containing a single-chain variable fragment derived from an anti-NKG2A antibody were linked to endoplasmic reticulum-retention domains. After retroviral transduction in human peripheral blood NK cells, these NKG2A Protein Expression Blockers (PEBLs) abrogated NKG2A expression. The resulting NKG2Anull NK cells had higher cytotoxicity against HLA-E-expressing tumor cells. Transduction of anti-NKG2A PEBL produced more potent cytotoxicity than interference with an anti-NKG2A antibody and prevented de novo NKG2A expression, without affecting NK cell proliferation. In immunodeficient mice, NKG2Anull NK cells were significantly more powerful than NKG2A+ NK cells against HLA-E-expressing tumors. Thus, NKG2A downregulation evades the HLA-E cancer immune-checkpoint, and increases the anti-tumor activity of NK cell infusions. Because this strategy is easily adaptable to current protocols for clinical-grade immune cell processing, its clinical testing is feasible and warranted.
Takahiro Kamiya, See Voon Seow, Desmond Wong, Murray Robinson, Dario Campana
Background: Systems vaccinology allows cutting-edge analysis of innate biomarkers of vaccine efficacy. We have been exploring novel strategies to shape the adaptive immune response, by targeting innate immune cells through novel immunization routes. Methods: This randomized phase I/II clinical study (n=60 healthy subjects aged 18-45 years old) used transcriptomic analysis to discover early biomarkers of immune response quality after transcutaneous (t.c.), intradermal (i.d.), and intramuscular (i.m.) administration of a trivalent influenza vaccine (TIV season 2012-2013) (1:1:1 ratio). Safety and immunogenicity (hemagglutinin inhibition (HI), microneutralization (MN) antibodies and CD4, CD8 effector T cells) were measured at baseline Day (D)0 and at D21. Blood transcriptome was analyzed at D0 and D1. Results: TIV-specific CD8+GranzymeB+(GRZ) T cells appeared in more individuals immunized by the t.c. and i.d. routes, while immunization by the i.d. and i.m. routes prompted high levels of HI antibody titers and MN against A/H1N1 and A/H3N2 influenza viral strains. The early innate gene signature anticipated immunological outcome by discriminating two clusters of individuals with either distinct humoral or CD8 cytotoxic responses. Several pathways explained this dichotomy confirmed by nine genes and serum level of CXCL10 were correlated with either TIV-specific cytotoxic CD8+GRZ+ T-cell or antibody responses. A logistic regression analysis demonstrated that these nine genes and serum levels of CXCL10 (D1/D0) best foreseen TIV-specific CD8+GRZ+ T-cell and antibody responses at D21. Conclusion: This study provides new insight into the impact of immunization routes and innate signature in the quality of adaptive immune responses.
Eléna Gonçalves, Olivia Bonduelle, Angèle Soria, Pierre Loulergue, Alexandra Rousseau, Marine Cachanado, Henri Bonnabau, Rodolphe Thiebaut, Nicolas Tchitchek, Sylvie Behillil, Sylvie van der Werf, Annika Vogt, Tabassome Simon, Odile Launay, Behazine Combadière
BRAF and CRAF are critical components of the MAPK signaling pathway which is activated in many cancer types. In approximately 1% of melanomas, BRAF or CRAF are activated through structural arrangements. We describe here a metastatic melanoma with a GOLGA4-RAF1 fusion and pathogenic variants in CTNNB1 and CDKN2A. Anti-CTLA4/anti-PD1 combination immunotherapy failed to control tumor progression. In the absence of other actionable variants the patient was administered MEK inhibitor therapy on the basis of its potential action against RAF1 fusions. This resulted in a profound and clinically significant response. We demonstrated that GOLGA4-RAF1 expression was associated with ERK activation, elevated expression of the RAS/RAF downstream co-effector ETV5, and a high Ki67 index. These findings provide a rationale for the dramatic response to targeted therapy. This study shows that thorough molecular characterization of treatment-resistant cancers can identify therapeutic targets and personalize management, leading to improved patient outcomes.
Christopher R. McEvoy, Huiling Xu, Kortnye Smith, Dariush Etemadmoghadam, Huei San Leong, David Y. Choong, David J. Byrne, Amir Iravani, Sophie Beck, Linda Mileshkin, Richard W. Tothill, David D. Bowtell, Bindi M. Bates, Violeta Nastevski, Judy Browning, Anthony H. Bell, Chloe Khoo, Jayesh Desai, Andrew P. Fellowes, Stephen B. Fox, Owen W.J. Prall
BACKGROUND. Recent genomic and bioinformatic technological advances have made it possible to dissect the immune response to personalized neoantigens encoded by tumor-specific mutations. However, timely and efficient identification of neoantigens is still one of the major obstacles to using personalized neoantigen-based cancer immunotherapy. METHODS. Two different pipelines of neoantigens identification were established in this study: (1) Clinical grade targeted sequencing was performed in patients with refractory solid tumor, and mutant peptides with high variant allele frequency and predicted high HLA-binding affinity were de novo synthesized. (2) An inventory-shared neoantigen peptide library of common solid tumors was constructed, and patients' hotspot mutations were matched to the neoantigen peptide library. The candidate neoepitopes were identified by recalling memory T-cell responses in vitro. Subsequently, neoantigen-loaded dendritic cell vaccines and neoantigen-reactive T cells were generated for personalized immunotherapy in six patients. RESULTS. Immunogenic neo-epitopes were recognized by autologous T cells in 3 of 4 patients who utilized the de novo synthesis mode and in 6 of 13 patients who performed shared neoantigen peptide library, respectively. A metastatic thymoma patient achieved a complete and durable response beyond 29 months after treatment. Immune-related partial response was observed in another patient with metastatic pancreatic cancer. The remaining four patients achieved the prolonged stabilization of disease with a median PFS of 8.6 months. CONCLUSIONS. The current study provided feasible pipelines for neoantigen identification. Implementing these strategies to individually tailor neoantigens could facilitate the neoantigen-based translational immunotherapy research. TRIAL REGSITRATION. ChiCTR.org ChiCTR-OIC-16010092, ChiCTR-OIC-17011275, ChiCTR-OIC-17011913; ClinicalTrials.gov NCT03171220. FUNDING. This work was funded by grants from the National Key Research and Development Program of China (Grant No. 2017YFC1308900), the National Major Projects for “Major New Drugs Innovation and Development” (Grant No.2018ZX09301048-003), the National Natural Science Foundation of China (Grant No. 81672367, 81572329, 81572601), and the Key Research and Development Program of Jiangsu Province (No. BE2017607).
Fangjun Chen, Zhengyun Zou, Juan Du, Shu Su, Jie Shao, Fanyan Meng, Ju Yang, Qiuping Xu, Naiqing Ding, Yang Yang, Qin Liu, Qin Wang, Zhichen Sun, Shujuan Zhou, Shiyao Du, Jia Wei, Baorui Liu
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
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.
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
Septic patients frequently develop cognitive impairment that persists beyond hospital discharge. The impact of sepsis on electrophysiological and molecular determinants of learning is underexplored. We observed that mouse survivors of sepsis or endotoxemia experienced loss of hippocampal long-term potentiation (LTP), a brain-derived neurotrophic factor (BDNF)-mediated process responsible for spatial memory formation. Memory impairment occurred despite preserved hippocampal BDNF content and could be reversed by stimulation of BDNF signaling, suggesting the presence of a local BDNF inhibitor. Sepsis is associated with degradation of the endothelial glycocalyx, releasing heparan sulfate fragments (of sufficient size and sulfation to bind BDNF) into the circulation. Heparan sulfate fragments penetrated the hippocampal blood-brain barrier during sepsis and inhibited BDNF-mediated LTP. Glycoarray approaches demonstrated that heparan sulfate’s avidity for BDNF increased with sulfation at the 2-O-position of iduronic acid and N-position of glucosamine. Circulating heparan sulfate in endotoxemic mice and septic humans was enriched in 2-O- and N-sulfated disaccharides; furthermore, the presence of these sulfation patterns in the plasma of septic patients at intensive care unit (ICU) admission predicted persistent cognitive impairment 14 days after ICU discharge or at hospital discharge. Our findings indicate that circulating 2-O- and N-sulfated heparan sulfate fragments contribute to septic cognitive impairment.
Joseph A. Hippensteel, Brian J. Anderson, James E. Orfila, Sarah A. McMurtry, Robert M. Dietz, Guowei Su, Joshay A. Ford, Kaori Oshima, Yimu Yang, Fuming Zhang, Xiaorui Han, Yanlei Yu, Jian Liu, Robert J. Linhardt, Nuala J. Meyer, Paco S. Herson, Eric P. Schmidt
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.
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
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.
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
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.
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