BACKGROUND. Liquid biopsies have demonstrated that the constitutively active androgen receptor splice variant-7 (AR-V7) associates with reduced response and overall survival from endocrine therapies in castration-resistant prostate cancer (CRPC). However, these studies provide little information pertaining to AR-V7 expression in prostate cancer (PC) tissue. METHODS. Following generation and validation of a potentially novel AR-V7 antibody for IHC, AR-V7 protein expression was determined for 358 primary prostate samples and 293 metastatic biopsies. Associations with disease progression, full-length androgen receptor (AR-FL) expression, response to therapy, and gene expression were determined. RESULTS. We demonstrated that AR-V7 protein is rarely expressed (<1%) in primary PC but is frequently detected (75% of cases) following androgen deprivation therapy, with further significant (P = 0.020) increase in expression following abiraterone acetate or enzalutamide therapy. In CRPC, AR-V7 expression is predominantly (94% of cases) nuclear and correlates with AR-FL expression (P ≤ 0.001) and AR copy number (P = 0.026). However, dissociation of expression was observed, suggesting that mRNA splicing remains crucial for AR-V7 generation. AR-V7 expression was heterogeneous between different metastases from a patient, although AR-V7 expression was similar within a metastasis. Moreover, AR-V7 expression correlated with a unique 59-gene signature in CRPC, including HOXB13, a critical coregulator of AR-V7 function. Finally, AR-V7–negative disease associated with better prostate-specific antigen (PSA) responses (100% vs. 54%, P = 0.03) and overall survival (74.3 vs. 25.2 months, hazard ratio 0.23 [0.07–0.79], P = 0.02) from endocrine therapies (pre-chemotherapy). CONCLUSION. This study provides impetus to develop therapies that abrogate AR-V7 signaling to improve our understanding of AR-V7 biology and to confirm the clinical significance of AR-V7. FUNDING. Work at the University of Washington and in the Plymate and Nelson laboratories is supported by the Department of Defense Prostate Cancer Research Program (W81XWH-14-2-0183, W81XWH-12-PCRP-TIA, W81XWH-15-1-0430, and W81XWH-13-2-0070), the Pacific Northwest Prostate Cancer SPORE (P50CA97186), the Institute for Prostate Cancer Research, the Veterans Affairs Research Program, the NIH/National Cancer Institute (P01CA163227), and the Prostate Cancer Foundation. Work in the de Bono laboratory was supported by funding from the Movember Foundation/Prostate Cancer UK (CEO13-2-002), the US Department of Defense (W81XWH-13-2-0093), the Prostate Cancer Foundation (20131017 and 20131017-1), Stand Up To Cancer (SU2C-AACR-DT0712), Cancer Research UK (CRM108X-A25144), and the UK Department of Health through an Experimental Cancer Medicine Centre grant (ECMC-CRM064X).
Adam Sharp, Ilsa Coleman, Wei Yuan, Cynthia Sprenger, David Dolling, Daniel Nava Rodrigues, Joshua W. Russo, Ines Figueiredo, Claudia Bertan, George Seed, Ruth Riisnaes, Takuma Uo, Antje Neeb, Jonathan Welti, Colm Morrissey, Suzanne Carreira, Jun Luo, Peter S. Nelson, Steven P. Balk, Lawrence D. True, Johann S. de Bono, Stephen R. Plymate
The negatively charged sugar sialic acid (Sia) occupies the outermost position in the bulk of cell surface glycans. Lack of sialylated glycans due to genetic ablation of the Sia-activating enzyme CMP–sialic acid synthase (CMAS) resulted in embryonic lethality around day 9.5 post coitum (E9.5) in mice. Developmental failure was caused by complement activation on trophoblasts in Cmas–/– implants and was accompanied by infiltration of maternal neutrophils at the fetal-maternal interface, intrauterine growth restriction, impaired placental development, and a thickened Reichert’s membrane. This phenotype, which shared features with complement receptor 1-related protein Y (Crry) depletion, was rescued in E8.5 Cmas–/– mice upon injection of cobra venom factor, resulting in exhaustion of the maternal complement component C3. Here we show that Sia is dispensable for early development of the embryo proper but pivotal for fetal-maternal immune homeostasis during pregnancy, i.e., for protecting the allograft implant against attack by the maternal innate immune system. Finally, embryos devoid of cell surface sialylation suffered from malnutrition due to inadequate placentation as a secondary effect.
Markus Abeln, Iris Albers, Ulrike Peters-Bernard, Kerstin Flächsig-Schulz, Elina Kats, Andreas Kispert, Stephen Tomlinson, Rita Gerardy-Schahn, Anja Münster-Kühnel, Birgit Weinhold
Iron-related disorders are among the most prevalent diseases worldwide. Systemic iron homeostasis requires hepcidin, a liver-derived hormone that controls iron mobilization through its molecular target ferroportin (FPN), the only known mammalian iron exporter. This pathway is perturbed in diseases that cause iron overload. Additionally, intestinal HIF-2α is essential for the local absorptive response to systemic iron deficiency and iron overload. Our data demonstrate a hetero-tissue crosstalk mechanism, whereby hepatic hepcidin regulated intestinal HIF-2α in iron deficiency, anemia, and iron overload. We show that FPN controlled cell-autonomous iron efflux to stabilize and activate HIF-2α by regulating the activity of iron-dependent intestinal prolyl hydroxylase domain enzymes. Pharmacological blockade of HIF-2α using a clinically relevant and highly specific inhibitor successfully treated iron overload in a mouse model. These findings demonstrate a molecular link between hepatic hepcidin and intestinal HIF-2α that controls physiological iron uptake and drives iron hyperabsorption during iron overload.
Andrew J. Schwartz, Nupur K. Das, Sadeesh K. Ramakrishnan, Chesta Jain, Mladen T. Jurkovic, Jun Wu, Elizabeta Nemeth, Samira Lakhal-Littleton, Justin A. Colacino, Yatrik M. Shah
Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent edema attacks associated with morbidity and mortality. HAE results from variations in the SERPING1 gene that encodes the C1 inhibitor (C1INH), a serine protease inhibitor (serpin). Reduced plasma levels of C1INH lead to enhanced activation of the contact system, triggering high levels of bradykinin and increased vascular permeability, but the cellular mechanisms leading to low C1INH levels (20%–30% of normal) in heterozygous HAE type I patients remain obscure. Here, we showed that C1INH encoded by a subset of HAE-causing SERPING1 alleles affected secretion of normal C1INH protein in a dominant-negative fashion by triggering formation of protein-protein interactions between normal and mutant C1INH, leading to the creation of larger intracellular C1INH aggregates that were trapped in the endoplasmic reticulum (ER). Notably, intracellular aggregation of C1INH and ER abnormality were observed in fibroblasts from a heterozygous carrier of a dominant-negative SERPING1 gene variant, but the condition was ameliorated by viral delivery of the SERPING1 gene. Collectively, our data link abnormal accumulation of serpins, a hallmark of serpinopathies, with dominant-negative disease mechanisms affecting C1INH plasma levels in HAE type I patients, and may pave the way for new treatments of HAE.
Didde Haslund, Laura Barrett Ryø, Sara Seidelin Majidi, Iben Rose, Kristian Alsbjerg Skipper, Tue Fryland, Anja Bille Bohn, Claus Koch, Martin K. Thomsen, Yaseelan Palarasah, Thomas J. Corydon, Anette Bygum, Lene N. Nejsum, Jacob Giehm Mikkelsen
Adoptive transfer of T cell receptor–engineered (TCR-engineered) T cells is a promising approach in cancer therapy but needs improvement for more effective treatment of solid tumors. While most clinical approaches have focused on CD8+ T cells, the importance of CD4+ T cells in mediating tumor regression has become apparent. Regarding shared (self) tumor antigens, it is unclear whether the human CD4+ T cell repertoire has been shaped by tolerance mechanisms and lacks highly functional TCRs suitable for therapy. Here, TCRs against the tumor-associated antigen NY-ESO-1 were isolated either from human CD4+ T cells or from mice that express a diverse human TCR repertoire with HLA-DRA/DRB1*0401 restriction and are NY-ESO-1 negative. NY-ESO-1–reactive TCRs from the mice showed superior recognition of tumor cells and higher functional activity compared with TCRs from humans. We identified a candidate TCR, TCR-3598_2, which was expressed in CD4+ T cells and caused tumor regression in combination with NY-ESO-1–redirected CD8+ T cells in a mouse model of adoptive T cell therapy. These data suggest that MHC II–restricted TCRs against NY-ESO-1 from a nontolerant nonhuman host are of optimal affinity and that the combined use of MHC I– and II–restricted TCRs against NY-ESO-1 can make adoptive T cell therapy more effective.
Lucia Poncette, Xiaojing Chen, Felix K.M. Lorenz, Thomas Blankenstein
BACKGROUND. Patients with schizophrenia (SCZ) experience chronic cognitive deficits. Histone deacetylases (HDACs) are enzymes that regulate cognitive circuitry; however, the role of HDACs in cognitive disorders, including SCZ, remains unknown in humans. We previously determined that HDAC2 mRNA levels were lower in dorsolateral prefrontal cortex (DLPFC) tissue from donors with SCZ compared with controls. Here we investigated the relationship between in vivo HDAC expression and cognitive impairment in patients with SCZ and matched healthy controls using [11C]Martinostat positron emission tomography (PET). METHODS. In a case-control study, relative [11C]Martinostat uptake was compared between 14 patients with SCZ or schizoaffective disorder (SCZ/SAD) and 17 controls using hypothesis-driven region-of-interest analysis and unbiased whole brain voxel-wise approaches. Clinical measures, including the MATRICS consensus cognitive battery, were administered. RESULTS. Relative HDAC expression was lower in the DLPFC of patients with SCZ/SAD compared with controls, and HDAC expression positively correlated with cognitive performance scores across groups. Patients with SCZ/SAD also showed lower relative HDAC expression in the dorsomedial prefrontal cortex and orbitofrontal gyrus, and higher relative HDAC expression in the cerebral white matter, pons, and cerebellum compared with controls. CONCLUSIONS. These findings provide in vivo evidence of HDAC dysregulation in patients with SCZ and suggest that altered HDAC expression may impact cognitive function in humans. FUNDING. National Institute of Mental Health (NIMH), Brain and Behavior Foundation, Massachusetts General Hospital (MGH), Athinoula A. Martinos Center for Biomedical Imaging, National Institute of Biomedical Imaging and Bioengineering (NIBIB), NIH Shared Instrumentation Grant Program.
Tonya M. Gilbert, Nicole R. Zürcher, Christine J. Wu, Anisha Bhanot, Baileigh G. Hightower, Minhae Kim, Daniel S. Albrecht, Hsiao-Ying Wey, Frederick A. Schroeder, Anais Rodriguez-Thompson, Thomas M. Morin, Kamber L. Hart, Amelia M. Pellegrini, Misha M. Riley, Changning Wang, Steven M. Stufflebeam, Stephen J. Haggarty, Daphne J. Holt, Marco L. Loggia, Roy H. Perlis, Hannah E. Brown, Joshua L. Roffman, Jacob M. Hooker
While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte–associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4–binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1–/– mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2–bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.
Chien-Chun Steven Pai, Donald M. Simons, Xiaoqing Lu, Michael Evans, Junnian Wei, Yung-hua Wang, Mingyi Chen, John Huang, Chanhyuk Park, Anthony Chang, Jiaxi Wang, Susan Westmoreland, Christine Beam, Dave Banach, Diana Bowley, Feng Dong, Jane Seagal, Wendy Ritacco, Paul L. Richardson, Soumya Mitra, Grace Lynch, Pete Bousquet, John Mankovich, Gillian Kingsbury, Lawrence Fong
Activation of the type 1 angiotensin II receptor (AT1) triggers proinflammatory signaling through pathways independent of classical Gq signaling that regulate vascular homeostasis. Here, we report that the AT1 receptor preformed a heteromeric complex with the receptor for advanced glycation endproducts (RAGE). Activation of the AT1 receptor by angiotensin II (Ang II) triggered transactivation of the cytosolic tail of RAGE and NF-κB–driven proinflammatory gene expression independently of the liberation of RAGE ligands or the ligand-binding ectodomain of RAGE. The importance of this transactivation pathway was demonstrated by our finding that adverse proinflammatory signaling events induced by AT1 receptor activation were attenuated when RAGE was deleted or transactivation of its cytosolic tail was inhibited. At the same time, classical homeostatic Gq signaling pathways were unaffected by RAGE deletion or inhibition. These data position RAGE transactivation by the AT1 receptor as a target for vasculoprotective interventions. As proof of concept, we showed that treatment with the mutant RAGE peptide S391A-RAGE362–404 was able to inhibit transactivation of RAGE and attenuate Ang II–dependent inflammation and atherogenesis. Furthermore, treatment with WT RAGE362–404 restored Ang II–dependent atherogenesis in Ager/Apoe-KO mice, without restoring ligand-mediated signaling via RAGE, suggesting that the major effector of RAGE activation was its transactivation.
Raelene J. Pickering, Christos Tikellis, Carlos J. Rosado, Despina Tsorotes, Alexandra Dimitropoulos, Monique Smith, Olivier Huet, Ruth M. Seeber, Rekhati Abhayawardana, Elizabeth K.M. Johnstone, Jonathan Golledge, Yutang Wang, Karin A. Jandeleit-Dahm, Mark E. Cooper, Kevin D.G. Pfleger, Merlin C. Thomas
Replicative immortality is a hallmark of cancer cells governed by telomere maintenance. Approximately 90% of human cancers maintain their telomeres by activating telomerase, driven by the transcriptional upregulation of telomerase reverse transcriptase (TERT). Although TERT promoter mutations (TPMs) are a major cancer-associated genetic mechanism of TERT upregulation, many cancers exhibit TERT upregulation without TPMs. In this study, we describe the TERT hypermethylated oncological region (THOR), a 433-bp genomic region encompassing 52 CpG sites located immediately upstream of the TERT core promoter, as a cancer-associated epigenetic mechanism of TERT upregulation. Unmethylated THOR repressed TERT promoter activity regardless of TPM status, and hypermethylation of THOR counteracted this repressive function. THOR methylation analysis in 1,352 human tumors revealed frequent (>45%) cancer-associated DNA hypermethylation in 9 of 11 (82%) tumor types screened. Additionally, THOR hypermethylation, either independently or along with TPMs, accounted for how approximately 90% of human cancers can aberrantly activate telomerase. Thus, we propose that THOR hypermethylation is a prevalent telomerase-activating mechanism in cancer that can act independently of or in conjunction with TPMs, further supporting the utility of THOR hypermethylation as a prognostic biomarker.
Donghyun D. Lee, Ricardo Leão, Martin Komosa, Marco Gallo, Cindy H. Zhang, Tatiana Lipman, Marc Remke, Abolfazl Heidari, Nuno Miguel Nunes, Joana D. Apolónio, Aryeh J. Price, Ramon Andrade De Mello, João S. Dias, David Huntsman, Thomas Hermanns, Peter J. Wild, Robert Vanner, Gelareh Zadeh, Jason Karamchandani, Sunit Das, Michael D. Taylor, Cynthia E. Hawkins, Jonathan D. Wasserman, Arnaldo Figueiredo, Robert J. Hamilton, Mark D. Minden, Khalida Wani, Bill Diplas, Hai Yan, Kenneth Aldape, Mohammad R. Akbari, Arnavaz Danesh, Trevor J. Pugh, Peter B. Dirks, Pedro Castelo-Branco, Uri Tabori
Acetaldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme detoxifying acetaldehyde and endogenous lipid aldehydes; previous studies suggest a protective role of ALDH2 against cardiovascular disease (CVD). Around 40% of East Asians carrying the single nucleotide polymorphism (SNP) ALDH2 rs671 have an increased incidence of CVD. However, the role of ALDH2 in CVD beyond alcohol consumption remains poorly defined. Here we report that ALDH2/LDLR double knockout (DKO) mice have decreased atherosclerosis compared with LDLR-KO mice, whereas ALDH2/APOE-DKO mice have increased atherosclerosis, suggesting an unexpected interaction of ALDH2 with LDLR. Further studies demonstrate that in the absence of LDLR, AMPK phosphorylates ALDH2 at threonine 356 and enables its nuclear translocation. Nuclear ALDH2 interacts with HDAC3 and represses transcription of a lysosomal proton pump protein ATP6V0E2, critical for maintaining lysosomal function, autophagy, and degradation of oxidized low-density lipid protein. Interestingly, an interaction of cytosolic LDLR C-terminus with AMPK blocks ALDH2 phosphorylation and subsequent nuclear translocation, whereas ALDH2 rs671 mutant in human macrophages attenuates this interaction, which releases ALDH2 to the nucleus to suppress ATP6V0E2 expression, resulting in increased foam cells due to impaired lysosomal function. Our studies reveal a novel role of ALDH2 and LDLR in atherosclerosis and provide a molecular mechanism by which ALDH2 rs671 SNP increases CVD.
Shanshan Zhong, Luxiao Li, Yu-Lei Zhang, Lili Zhang, Jianhong Lu, Shuyuan Guo, Ningning Liang, Jing Ge, Mingjiang Zhu, Yongzhen Tao, Yun-Cheng Wu, Huiyong Yin