Hepatocellular carcinoma (HCC) is a leading cause of death among cirrhotic patients, for which chemopreventive strategies are lacking. Recently, we developed a simple human cell-based system modeling a clinical prognostic liver signature (PLS) predicting liver disease progression and HCC risk. In a previous study, we applied our cell-based system for drug discovery and identified captopril, an approved angiotensin converting enzyme (ACE) inhibitor, as a candidate compound for HCC chemoprevention. Here, we explored ACE as a therapeutic target for HCC chemoprevention. Captopril reduced liver fibrosis and effectively prevented liver disease progression toward HCC development in a diethylnitrosamine (DEN) rat cirrhosis model and a diet-based rat model for nonalcoholic steatohepatitis–induced (NASH-induced) hepatocarcinogenesis. RNA-Seq analysis of cirrhotic rat liver tissues uncovered that captopril suppressed the expression of pathways mediating fibrogenesis, inflammation, and carcinogenesis, including epidermal growth factor receptor (EGFR) signaling. Mechanistic data in liver disease models uncovered a cross-activation of the EGFR pathway by angiotensin. Corroborating the clinical translatability of the approach, captopril significantly reversed the HCC high-risk status of the PLS in liver tissues of patients with advanced fibrosis. Captopril effectively prevents fibrotic liver disease progression toward HCC development in preclinical models and is a generic and safe candidate drug for HCC chemoprevention.
Emilie Crouchet, Shen Li, Mozhdeh Sojoodi, Simonetta Bandiera, Naoto Fujiwara, Hussein El Saghire, Shijia Zhu, Tongqi Qian, Fahmida Akter Rasha, Fabio Del Zompo, Stephen C. Barrett, Eugénie Schaeffer, Marine A. Oudot, Clara Ponsolles, Sarah C. Durand, Sarani Ghoshal, Gunisha Arora, Fabio Giannone, Raymond T. Chung, Nevena Slovic, Nicolaas Van Renne, Emanuele Felli, Patrick Pessaux, Joachim Lupberger, Nathalie Pochet, Catherine Schuster, Kenneth K. Tanabe, Yujin Hoshida, Bryan C. Fuchs, Thomas F. Baumert
Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin with 2 etiologies. Merkel cell polyomavirus (MCPyV) integration is present in about 80% of all MCC. Virus-positive MCC (MCCP) tumors have few somatic mutations and usually express WT p53 (TP53). By contrast, virus-negative MCC (MCCN) tumors present with a high tumor mutational burden and predominantly UV mutational signature. MCCN tumors typically contain mutated TP53. MCCP tumors express 2 viral proteins: MCPyV small T antigen and a truncated form of large T antigen. MCPyV ST specifically activates expression of MDM2, an E3 ubiquitin ligase of p53, to inhibit p53-mediated tumor suppression. In this study, we assessed the efficacy of milademetan, a potent, selective, and orally available MDM2 inhibitor in several MCC models. Milademetan reduced cell viability of WT p53 MCC cell lines and triggered a rapid and sustained p53 response. Milademetan showed a dose-dependent inhibition of tumor growth in MKL-1 xenograft and patient-derived xenograft models. Here, along with preclinical data for the efficacy of milademetan in WT p53 MCC tumors, we report several in vitro and in vivo models useful for future MCC studies.
Varsha Ananthapadmanabhan, Thomas C. Frost, Kara M. Soroko, Aine Knott, Brianna J. Magliozzi, Prafulla C. Gokhale, Vijaya G. Tirunagaru, Robert C. Doebele, James A. DeCaprio
Cyclophosphamide (CPA) and doxorubicin (DOX) are key components of chemotherapy for triple-negative breast cancer (TNBC), although suboptimal outcomes are commonly associated with drug resistance and/or intolerable side effects. Through an approach combining high-throughput screening and chemical modification, we developed CN06 as a dual activator of the constitutive androstane receptor (CAR) and nuclear factor erythroid 2-related factor 2 (Nrf2). CN06 enhances CAR-induced bioactivation of CPA (a prodrug) by provoking hepatic expression of CYP2B6, while repressing DOX-induced cytotoxicity in cardiomyocytes in vitro via stimulating Nrf2-antioxidant signaling. Utilizing a multicellular coculture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we show that CN06 increased CPA/DOX-mediated TNBC cell death via CAR-dependent CYP2B6 induction and subsequent conversion of CPA to its active metabolite 4-hydroxy-CPA, while protecting against DOX-induced cardiotoxicity by selectively activating Nrf2-antioxidant signaling in cardiomyocytes but not in TNBC cells. Furthermore, CN06 preserves the viability and function of human iPSC–derived cardiomyocytes by modulating antioxidant defenses, decreasing apoptosis, and enhancing the kinetics of contraction and relaxation. Collectively, our findings identify CAR and Nrf2 as potentially novel combined therapeutic targets whereby CN06 holds the potential to improve the efficacy/toxicity ratio of CPA/DOX-containing chemotherapy.
Sydney Stern, Dongdong Liang, Linhao Li, Ritika Kurian, Caitlin Lynch, Srilatha Sakamuru, Scott Heyward, Junran Zhang, Kafayat Ajoke Kareem, Young Wook Chun, Ruili Huang, Menghang Xia, Charles C. Hong, Fengtian Xue, Hongbing Wang
Nemaline myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle–specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as cap myopathy, congenital fiber type disproportion, and distal arthrogryposis (DA). The in vivo pathomechanisms underlying TPM2-related disorders are unknown, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found 4 variants significantly affected muscle development and muscle function. Transient overexpression of the 4 variants also disrupted the morphogenesis of mouse myotubes in vitro and negatively affected zebrafish muscle development in vivo. We used transient overexpression assays in zebrafish to characterize 2 potentially novel TPM2 variants and 1 recurring variant that we identified in patients with DA (V129A, E139K, A155T, respectively) and found these variants caused musculoskeletal defects similar to those of known pathogenic variants. The consistency of musculoskeletal phenotypes in our assays correlated with the severity of clinical phenotypes observed in our patients with DA, suggesting disrupted myogenesis is a potentially novel pathomechanism of TPM2 disorders and that our myogenic assays can predict the clinical severity of TPM2 variants.
Jennifer McAdow, Shuo Yang, Tiffany Ou, Gary Huang, Matthew B. Dobbs, Christina A. Gurnett, Michael J. Greenberg, Aaron N. Johnson
Recent studies highlighted the clinicopathologic importance of the tumor microenvironment (TME) in delineating molecular attributes and therapeutic potentials. However, the overall TME cell infiltration landscape in nonsquamous non–small cell lung cancer (NSCLC) has not been comprehensively characterized. In this study, we used consensus non-negative matrix factorization molecular subtyping to determine TME cell infiltration patterns and identified 3 TME clusters (TME-C1, -C2, -C3) characterized by distinct clinicopathologic features, infiltrating cells, and biological processes. Proteomics analyses revealed that cyclic GMP-AMP–stimulator of interferon genes immune signaling–mediated protein and phosphorylation levels were significantly upregulated in inflammation-related TME-C2 clusters. The score extracted from the TME-related signature (TMEsig-score) divided patients with NSCLC into high- and low-score subgroups, where a high score was associated with favorable prognosis and immune infiltration. The genomic landscape revealed that patients with low TMEsig-score harbored more somatic copy number alterations and higher mutation frequency of driver genes involving STK11, KEAP1, SMARCA4, and others. Drug sensitivity analyses suggested that tumors with high TMEsig-score were responsible for favorable clinical response to immune checkpoint inhibitor treatment. In summary, this study highlights that comprehensive recognizing of the TME cell infiltration landscape will contribute to enhancing our understanding of TME immune regulation and promote effectiveness of precision biotherapy strategies.
Hao Chen, Tongchao Zhang, Yuan Zhang, Hao Wu, Zhen Fang, Yang Liu, Yang Chen, Zhe Wang, Shengtao Jia, Xingzhao Ji, Liang Shang, Fengying Du, Jin Liu, Ming Lu, Wei Chong
In situ vaccination has demonstrated the feasibility of priming local immunity for systemic antitumor responses. Although direct intratumoral (IT) delivery of adjuvant is the mainstay, tumor-draining lymph nodes (TDLNs) also play essential roles in antitumor immunity. We report that directing an adjuvant to both tumors and TDLNs during in situ vaccination can induce robust antitumor responses. Conventional IT dosing leads to tumor-limited delivery of agents; however, delivery to both tumors and TDLNs can be ensured through a micellar formation. The peritumoral delivery of micellar MEDI9197 (mcMEDI), a toll-like receptor 7/8 agonist, induced significantly stronger innate and adaptive immune responses than those on conventional dosing. Optimal dosing was crucial because excessive or insufficient accumulation of the adjuvant in the TDLNs compromised therapeutic efficacy. The combination of local mcMEDI therapy significantly improved the efficacy of systemic anti–programmed death receptor 1 therapy. These data suggest that rerouting adjuvants to tumors and TDLNs can augment the therapeutic efficacy of in situ vaccination.
Moonkyoung Jeong, Heegon Kim, Junyong Yoon, Dong-Hyun Kim, Ji-Ho Park
People living with multiple sclerosis (MS) experience episodic CNS white matter lesions instigated by autoreactive T cells. With age, patients with MS show evidence of gray matter demyelination and experience devastating nonremitting symptomology. What drives progression is unclear and studying this has been hampered by the lack of suitable animal models. Here, we show that passive experimental autoimmune encephalomyelitis (EAE) induced by an adoptive transfer of young Th17 cells induced a nonremitting clinical phenotype that was associated with persistent leptomeningeal inflammation and cortical pathology in old, but not young, SJL/J mice. Although the quantity and quality of T cells did not differ in the brains of old versus young EAE mice, an increase in neutrophils and a decrease in B cells were observed in the brains of old mice. Neutrophils were also found in the leptomeninges of a subset of progressive MS patient brains that showed evidence of leptomeningeal inflammation and subpial cortical demyelination. Taken together, our data show that while Th17 cells initiate CNS inflammation, subsequent clinical symptoms and gray matter pathology are dictated by age and associated with other immune cells, such as neutrophils.
Michelle Zuo, Naomi M. Fettig, Louis-Philippe Bernier, Elisabeth Pössnecker, Shoshana Spring, Annie Pu, Xianjie I. Ma, Dennis S.W. Lee, Lesley A. Ward, Anshu Sharma, Jens Kuhle, John G. Sled, Anne-Katrin Pröbstel, Brian A. MacVicar, Lisa C. Osborne, Jennifer L. Gommerman, Valeria Ramaglia
Novel therapeutic strategies targeting glioblastoma (GBM) often fail in the clinic, partly because preclinical models in which hypotheses are being tested do not recapitulate human disease. To address this challenge, we took advantage of our previously developed spontaneous Qk/Trp53/Pten (QPP) triple-knockout model of human GBM, comparing the immune microenvironment of QPP mice with that of patient-derived tumors to determine whether this model provides opportunity for gaining insights into tumor physiopathology and preclinical evaluation of therapeutic agents. Immune profiling analyses and single-cell sequencing of implanted and spontaneous tumors from QPP mice and from patients with glioma revealed intratumoral immune components that were predominantly myeloid cells (e.g., monocytes, macrophages, and microglia), with minor populations of T, B, and NK cells. When comparing spontaneous and implanted mouse samples, we found more neutrophils and T and NK cells in the implanted model. Neutrophils and T and NK cells were increased in abundance in samples derived from human high-grade glioma compared with those derived from low-grade glioma. Overall, our data demonstrate that our implanted and spontaneous QPP models recapitulate the immunosuppressive myeloid-dominant nature of the tumor microenvironment of human gliomas. Our model provides a suitable tool for investigating the complex immune compartment of gliomas.
Daniel B. Zamler, Takashi Shingu, Laura M. Kahn, Kristin Huntoon, Cynthia Kassab, Martina Ott, Katarzyna Tomczak, Jintan Liu, Yating Li, Ivy Lai, Rocio Zorilla-Veloz, Cassian Yee, Kunal Rai, Betty Y.S. Kim, Stephanie S. Watowich, Amy B. Heimberger, Giulio F. Draetta, Jian Hu
Biological aging is the strongest factor associated with the clinical phenotype of multiple sclerosis (MS). Relapsing-remitting MS typically presents in the third or fourth decade, whereas the mean age of presentation of progressive MS (PMS) is 45 years old. Here, we show that experimental autoimmune encephalomyelitis (EAE), induced by the adoptive transfer of encephalitogenic CD4+ Th17 cells, was more severe, and less likely to remit, in middle-aged compared with young adult mice. Donor T cells and neutrophils were more abundant, while B cells were relatively sparse, in CNS infiltrates of the older mice. Experiments with reciprocal bone marrow chimeras demonstrated that radio-resistant, nonhematopoietic cells played a dominant role in shaping age-dependent features of the neuroinflammatory response, as well as the clinical course, during EAE. Reminiscent of PMS, EAE in middle-aged adoptive transfer recipients was characterized by widespread microglial activation. Microglia from older mice expressed a distinctive transcriptomic profile suggestive of enhanced chemokine synthesis and antigen presentation. Collectively, our findings suggest that drugs that suppress microglial activation, and acquisition or expression of aging-associated properties, may be beneficial in the treatment of progressive forms of inflammatory demyelinating disease.
Jeffrey R. Atkinson, Andrew D. Jerome, Andrew R. Sas, Ashley Munie, Cankun Wang, Anjun Ma, William D. Arnold, Benjamin M. Segal
Macrophages play a crucial role in the inflammatory response to the human stomach pathogen Helicobacter pylori, which infects half of the world’s population and causes gastric cancer. Recent studies have highlighted the importance of macrophage immunometabolism in their activation state and function. We have demonstrated that the cysteine-producing enzyme cystathionine γ-lyase (CTH) is upregulated in humans and mice with H. pylori infection. Here, we show that induction of CTH in macrophages by H. pylori promoted persistent inflammation. Cth–/– mice had reduced macrophage and T cell activation in H. pylori–infected tissues, an altered metabolome, and decreased enrichment of immune-associated gene networks, culminating in decreased H. pylori–induced gastritis. CTH is downstream of the proposed antiinflammatory molecule, S-adenosylmethionine (SAM). Whereas Cth–/– mice exhibited gastric SAM accumulation, WT mice treated with SAM did not display protection against H. pylori–induced inflammation. Instead, we demonstrated that Cth-deficient macrophages exhibited alterations in the proteome, decreased NF-κB activation, diminished expression of macrophage activation markers, and impaired oxidative phosphorylation and glycolysis. Thus, through altering cellular respiration, CTH is a key enhancer of macrophage activation, contributing to a pathogenic inflammatory response that is the universal precursor for the development of H. pylori–induced gastric disease.
Yvonne L. Latour, Johanna C. Sierra, Jordan L. Finley, Mohammad Asim, Daniel P. Barry, Margaret M. Allaman, Thaddeus M. Smith, Kara M. McNamara, Paula B. Luis, Claus Schneider, Justin Jacobse, Jeremy A. Goettel, M. Wade Calcutt, Kristie L. Rose, Kevin L. Schey, Ginger L. Milne, Alberto G. Delgado, M. Blanca Piazuelo, Bindu D. Paul, Solomon H. Snyder, Alain P. Gobert, Keith T. Wilson
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