Research Article
Abstract
Accurately identifying patients who respond to immunotherapy remains clinically challenging. A noninvasive method that can longitudinally capture information about immune cell function and assist in the early assessment of tumor responses is highly desirable for precision immunotherapy. Here, we show that PET imaging using a granzyme B–targeted radiotracer named 68Ga-grazytracer, could noninvasively and effectively predict tumor responses to immune checkpoint inhibitors and adoptive T cell transfer therapy in multiple tumor models. 68Ga-grazytracer was designed and selected from several radiotracers based on non-aldehyde peptidomimetics, and exhibited excellent in vivo metabolic stability and favorable targeting efficiency to granzyme B secreted by effector CD8+ T cells during immune responses. 68Ga-grazytracer permitted more sensitive discrimination of responders and nonresponders than did 18F-fluorodeoxyglucose, distinguishing between tumor pseudoprogression and true progression upon immune checkpoint blockade therapy in mouse models with varying immunogenicity. In a preliminary clinical trial with 5 patients, no adverse events were observed after 68Ga-grazytracer injection, and clinical responses in cancer patients undergoing immunotherapy were favorably correlated with 68Ga-grazytracer PET results. These results highlight the potential of 68Ga-grazytracer PET to enhance the clinical effectiveness of granzyme B secretion–related immunotherapies by supporting early response assessment and precise patient stratification in a noninvasive and longitudinal manner.
Authors
Haoyi Zhou, Yanpu Wang, Hongchuang Xu, Xiuling Shen, Ting Zhang, Xin Zhou, Yuwen Zeng, Kui Li, Li Zhang, Hua Zhu, Xing Yang, Nan Li, Zhi Yang, Zhaofei Liu
Abstract
Plasmacytoid dendritic cells (pDCs) are a professional type I IFN producer that play critical roles in the pathogenesis of autoimmune diseases. However, both genetic regulation of the function of pDCs and their relationships with autoimmunity are largely undetermined. Here, we investigated the causality of the neutrophil cytosolic factor 1 (NCF1) missense variant, which is one of the most significant associated risk variants for lupus, and found that the substitution of arginine (R) for histidine (H) at position 90 in the NCF1 protein (NCF1 p.R90H) led to excessive activation of pDCs. A mechanism study demonstrated that p.R90H reduced the affinity of NCF1 for phospholipids, thereby impairing endosomal localization of NCF1. As NCF1 is a subunit of the NADPH oxidase 2 (NOX2) complex, this impairment led to an acidified endosomal pH and facilitated downstream TLR signaling. Consistently, the homozygous knockin mice manifested aggravated lupus progression in a pDC-dependent lupus model. More important, pharmaceutical intervention revealed that hydroxychloroquine (HCQ) could antagonize the detrimental function of NCF1 p.R90H in the lupus model and systemic lupus erythematosus samples, supporting the idea that NCF1 p.R90H could be identified as a genetic biomarker for HCQ application. Therefore, our study provides insights into the genetic control of pDC function and a paradigm for applying genetic variants to improve targeted therapy for autoimmune diseases.
Authors
Yao Meng, Jianyang Ma, Chao Yao, Zhizhong Ye, Huihua Ding, Can Liu, Jun Li, Guanhua Li, Yuke He, Jia Li, Zhihua Yin, Li Wu, Haibo Zhou, Nan Shen
Abstract
Aberrant expression of viral-like repeat elements is a common feature of epithelial cancers, and the substantial diversity of repeat species provides a distinct view of the cancer transcriptome. Repeatome profiling across ovarian, pancreatic, and colorectal cell lines identifies distinct clustering independent of tissue origin that is seen with coding gene analysis. Deeper analysis of ovarian cancer cell lines demonstrated that human satellite II (HSATII) satellite repeat expression was highly associated with epithelial-mesenchymal transition (EMT) and anticorrelated with IFN-response genes indicative of a more aggressive phenotype. SATII expression — and its correlation with EMT and anticorrelation with IFN-response genes — was also found in ovarian cancer RNA-Seq data and was associated with significantly shorter survival in a second independent cohort of patients with ovarian cancer. Repeat RNAs were enriched in tumor-derived extracellular vesicles capable of stimulating monocyte-derived macrophages, demonstrating a mechanism that alters the tumor microenvironment with these viral-like sequences. Targeting of HSATII with antisense locked nucleic acids stimulated IFN response and induced MHC I expression in ovarian cancer cell lines, highlighting a potential strategy of modulating the repeatome to reestablish antitumor cell immune surveillance.
Authors
Rebecca L. Porter, Siyu Sun, Micayla N. Flores, Emily Berzolla, Eunae You, Ildiko E. Phillips, Neelima KC, Niyati Desai, Eric C. Tai, Annamaria Szabolcs, Evan R. Lang, Amaya Pankaj, Michael J. Raabe, Vishal Thapar, Katherine H. Xu, Linda T. Nieman, Daniel C. Rabe, David L. Kolin, Elizabeth H. Stover, David Pepin, Shannon L. Stott, Vikram Deshpande, Joyce F. Liu, Alexander Solovyov, Ursula A. Matulonis, Benjamin D. Greenbaum, David T. Ting
Abstract
Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR/Cas9 screens implicated decreased translation initiation as protective following GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to the effects of GSPT1 degradation. We defined 2 Crbn amino acids that prevent Gspt1 degradation in mice, generated a knockin mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant acute myeloid leukemia.
Authors
Rob S. Sellar, Adam S. Sperling, Mikołaj Słabicki, Jessica A. Gasser, Marie E. McConkey, Katherine A. Donovan, Nada Mageed, Dylan N. Adams, Charles Zou, Peter G. Miller, Ravi K. Dutta, Steffen Boettcher, Amy E. Lin, Brittany Sandoval, Vanessa A. Quevedo Barrios, Veronica Kovalcik, Jonas Koeppel, Elizabeth K. Henderson, Emma C. Fink, Lu Yang, Anthony Chan, Sheela Pangeni Pokharel, Erik J. Bergstrom, Rajan Burt, Namrata D. Udeshi, Steven A. Carr, Eric S. Fischer, Chun-Wei Chen, Benjamin L. Ebert
Abstract
Chimeric antigen receptor (CAR) T cell therapies targeting single antigens have performed poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T cell persistence, and T cell exhaustion. Here, we aimed to identify optimal CARs against glypican 2 (GPC2) or CD276 (B7-H3), which were highly but heterogeneously expressed in neuroblastoma (NB), a lethal extracranial solid tumor of childhood. First, we examined CAR T cell expansion in the presence of targets by digital droplet PCR. Next, using pooled competitive optimization of CAR by cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), termed P-COCC, we simultaneously analyzed protein and transcriptome expression of CAR T cells to identify high-activity CARs. Finally, we performed cytotoxicity assays to identify the most effective CAR against each target and combined the CARs into a bicistronic “OR” CAR (BiCisCAR). BiCisCAR T cells effectively eliminated tumor cells expressing GPC2 or CD276. Furthermore, the BiCisCAR T cells demonstrated prolonged persistence and resistance to exhaustion when compared with CARs targeting a single antigen. This study illustrated that targeting multiple TAAs with BiCisCAR may overcome heterogenous expression of target antigens in solid tumors and identified a potent, clinically relevant CAR against NB. Moreover, our multimodal approach integrating competitive expansion, P-COCC, and cytotoxicity assays is an effective strategy to identify potent CARs among a pool of candidates.
Authors
Meijie Tian, Adam T. Cheuk, Jun S. Wei, Abdalla Abdelmaksoud, Hsien-Chao Chou, David Milewski, Michael C. Kelly, Young K. Song, Christopher M. Dower, Nan Li, Haiying Qin, Yong Yean Kim, Jerry T. Wu, Xinyu Wen, Mehdi Benzaoui, Katherine E. Masih, Xiaolin Wu, Zhongmei Zhang, Sherif Badr, Naomi Taylor, Brad St. Croix, Mitchell Ho, Javed Khan
Abstract
Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.
Authors
Evan R. Abt, Khalid Rashid, Thuc M. Le, Suwen Li, Hailey R. Lee, Vincent Lok, Luyi Li, Amanda L. Creech, Amanda N. Labora, Hanna K. Mandl, Alex K. Lam, Arthur Cho, Valerie Rezek, Nanping Wu, Gabriel Abril-Rodriguez, Ethan W. Rosser, Steven D. Mittelman, Willy Hugo, Thomas Mehrling, Shanta Bantia, Antoni Ribas, Timothy R. Donahue, Gay M. Crooks, Ting-Ting Wu, Caius G. Radu
Abstract
With an aging population, kidney health becomes an important medical and socioeconomic factor. Kidney aging mechanisms are not well understood. We previously showed that podocytes isolated from aged mice exhibit increased expression of programmed cell death protein 1 (PD-1) surface receptor and its 2 ligands (PD-L1 and PD-L2). PDCD1 transcript increased with age in microdissected human glomeruli, which correlated with lower estimated glomerular filtration rate and higher segmental glomerulosclerosis and vascular arterial intima-to-lumen ratio. In vitro studies in podocytes demonstrated a critical role for PD-1 signaling in cell survival and in the induction of a senescence-associated secretory phenotype. To prove PD-1 signaling was critical to podocyte aging, aged mice were injected with anti–PD-1 antibody. Treatment significantly improved the aging phenotype in both kidney and liver. In the glomerulus, it increased the life span of podocytes, but not that of parietal epithelial, mesangial, or endothelial cells. Transcriptomic and immunohistochemistry studies demonstrated that anti–PD-1 antibody treatment improved the health span of podocytes. Administering the same anti–PD-1 antibody to young mice with experimental focal segmental glomerulosclerosis (FSGS) lowered proteinuria and improved podocyte number. These results suggest a critical contribution of increased PD-1 signaling toward both kidney and liver aging and in FSGS.
Authors
Jeffrey W. Pippin, Natalya Kaverina, Yuliang Wang, Diana G. Eng, Yuting Zeng, Uyen Tran, Carol J. Loretz, Anthony Chang, Shreeram Akilesh, Chetan Poudel, Hannah S. Perry, Christopher O’Connor, Joshua C. Vaughan, Markus Bitzer, Oliver Wessely, Stuart J. Shankland
Abstract
Fanconi anemia (FA) is the most prevalent inherited bone marrow failure (BMF) syndrome. Nevertheless, the pathophysiological mechanisms of BMF in FA have not been fully elucidated. Since FA cells are defective in DNA repair, we hypothesized that FA hematopoietic stem and progenitor cells (HSPCs) might express DNA damage–associated stress molecules such as natural killer group 2 member D ligands (NKG2D-Ls). These ligands could then interact with the activating NKG2D receptor expressed in cytotoxic NK or CD8+ T cells, which may result in progressive HSPC depletion. Our results indeed demonstrated upregulated levels of NKG2D-Ls in cultured FA fibroblasts and T cells, and these levels were further exacerbated by mitomycin C or formaldehyde. Notably, a high proportion of BM CD34+ HSPCs from patients with FA also expressed increased levels of NKG2D-Ls, which correlated inversely with the percentage of CD34+ cells in BM. Remarkably, the reduced clonogenic potential characteristic of FA HSPCs was improved by blocking NKG2D–NKG2D-L interactions. Moreover, the in vivo blockage of these interactions in a BMF FA mouse model ameliorated the anemia in these animals. Our study demonstrates the involvement of NKG2D–NKG2D-L interactions in FA HSPC functionality, suggesting an unexpected role of the immune system in the progressive BMF that is characteristic of FA.
Authors
José A. Casado, Antonio Valeri, Rebeca Sanchez-Domínguez, Paula Vela, Andrea López, Susana Navarro, Omaira Alberquilla, Helmut Hanenberg, Roser Pujol, José-Carlos Segovia, Jordi Minguillón, Jordi Surrallés, Cristina Díaz de Heredia, Julián Sevilla, Paula Rio, Juan A. Bueren
Abstract
Acquired resistance is inevitable in non–small cell lung cancers (NSCLCs) treated with osimertinib (OSI), and the mechanisms are not well defined. The MERTK ligand GAS6 promoted downstream oncogenic signaling in EGFR-mutated (EGFRMT) NSCLC cells treated with OSI, suggesting a role for MERTK activation in OSI resistance. Indeed, treatment with MRX-2843, a first-in-class MERTK kinase inhibitor, resensitized GAS6-treated NSCLC cells to OSI. Both GAS6 and EGF stimulated downstream PI3K/AKT and MAPK/ERK signaling in parental cells, but only GAS6 activated these pathways in OSI-resistant (OSIR) derivative cell lines. Functionally, OSIR cells were more sensitive to MRX-2843 than parental cells, suggesting acquired dependence on MERTK signaling. Furthermore, MERTK and/or its ligands were dramatically upregulated in EGFRMT tumors after treatment with OSI in both xenograft models and patient samples, consistent with induction of autocrine/paracrine MERTK activation. Moreover, treatment with MRX-2843 in combination with OSI, but not OSI alone, provided durable suppression of tumor growth in vivo, even after treatment was stopped. These data identify MERTK as a driver of bypass signaling in treatment-naive and EGFRMT-OSIR NSCLC cells and predict that MRX-2843 and OSI combination therapy will provide clinical benefit in patients with EGFRMT NSCLC.
Authors
Dan Yan, Justus M. Huelse, Dmitri Kireev, Zikang Tan, Luxiao Chen, Subir Goyal, Xiaodong Wang, Stephen V. Frye, Madhusmita Behera, Frank Schneider, Suresh S. Ramalingam, Taofeek Owonikoko, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham
Abstract
A prophylactic hepatitis C virus (HCV) vaccine that elicits neutralizing antibodies could be key to HCV eradication. However, the genetic and antigenic properties of HCV envelope (E1E2) proteins capable of inducing anti-HCV broadly neutralizing antibodies (bNAbs) in humans have not been defined. Here, we investigated the development of bNAbs in longitudinal plasma of HCV-infected persons with persistent infection or spontaneous clearance of multiple reinfections. By measuring plasma antibody neutralization of a heterologous virus panel, we found that the breadth and potency of the antibody response increased upon exposure to multiple genetically distinct infections and with longer duration of viremia. Greater genetic divergence between infecting strains was not associated with enhanced neutralizing breadth. Rather, repeated exposure to antigenically related, antibody-sensitive E1E2s was associated with potent bNAb induction. These data reveal that a prime-boost vaccine strategy with genetically distinct, antibody-sensitive viruses is a promising approach to inducing potent bNAbs in humans.
Authors
Nicole Frumento, Alexis Figueroa, Tingchang Wang, Muhammad N. Zahid, Shuyi Wang, Guido Massaccesi, Georgia Stavrakis, James E. Crowe Jr, Andrew I. Flyak, Hongkai Ji, Stuart C. Ray, George M. Shaw, Andrea L. Cox, Justin R. Bailey
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ISSN: 0021-9738 (print), 1558-8238 (online)