Signal Transduction and Targeted Therapy

Gut microbiota metabolic remodeling is a pivotal determinant in irinotecan-induced enterotoxicity and epithelial damage, although the underlying mechanisms remain unclear. Herein, we discovered that Daikenchuto (DKT), a traditional Chinese prescription for intestinal disorders, alleviated irinotecan-induced enterotoxicity without compromising its anti-tumor efficacy by improving weight loss, diarrhea, intestinal inflammation, and barrier damage, and these effects were partially dependent on gut microbiota. DKT significantly restored microbial tryptophan metabolism in irinotecan-treated rats, which was characterized by the enrichment of Limosilactobacillus reuteri, and elevated levels of indole-3-ethanol (IE) and indole-3-propionic acid (IPA). Multi-omics analysis further revealed a positive correlation between L. reuteri and IE and IPA. Consistent with this, DKT promoted L. reuteri proliferation, leading to the conversion of tryptophan to IE and IPA, which improved epithelial barrier damage in the irinotecan-treated Caco-2 cells. In addition, DKT suppressed the growth of Loop 1 {beta}-glucuronidase ({beta}-GUS)-producing bacteria, such as Escherichia coli. Furthermore, the main constituents of DKT selectively inhibited Loop 1 {beta}-GUS activity independent of the gut microbiota, which reduced the intra-luminal level of 7-ethyl-10-hydroxycamptothecin, the toxic metabolite of irinotecan. Taken together, this study reveals a dual gut microbiota-driven mechanism by which DKT mitigates irinotecan-induced enterotoxicity, which provides a promising strategy for managing chemotherapy-related enterotoxicity.

African swine fever (ASF) is a highly pathogenic disease caused by the African swine fever virus (ASFV) infection, which can affect pigs of all ages and breeds, posing significant threat to the global pig farming industry. The ASFV p30 protein is an early-expressed viral structural protein; however, its function is not fully understood. In this study, the interaction of viral p30 with host TRIM21 was identified. The ectopic TRIM21 inhibited ASFV replication, while knockdown or knockout of TRIM21 promoted ASFV replication. Further, p30 was found to interact with RIG-I-like receptor (RLR) signaling adaptor MAVS, and during ASFV infection, p30-TRIM21-MAVS interacted with each other. Mechanistically, TRIM21 activated the K27 polyubiquitination of MAVS to induce IRF3 mediated type I interferon (IFN) production, whereas p30 counteracted TRIM21 activated MAVS K27 polyubiquitination to evade RLR signaling mediated antiviral IFN induction. In summary, our study revealed a novel function of ASFV p30, and provided new insights into the immune evasion of ASFV.

Orthohantaviruses cause severe human diseases including hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with case fatality rates up to 40%. No FDA-approved therapeutics are currently available, highlighting urgent need for drug development following recent outbreak events. We systematically examined host protease dependencies in hantavirus replication, focusing on Signal Peptidase (SP) and Signal Peptide Peptidase (SPP) essential for viral glycoprotein maturation. Through comprehensive database mining and molecular docking analysis, we identified six potential protease inhibitors, with Compound E achieving the highest binding confidence score (-0.28) against SPP. Our analysis reveals that targeting host ER proteases represents a viable antiviral strategy, providing a systematic framework for protease-targeted antihantavirus drug development and identifying specific lead compounds for experimental validation.

Aberrant activation of type I interferon (IFN-I) is closely related to the development of autoimmune diseases. The metabolic regulation of cytokine signaling is essential for immune homeostasis. In this study, we characterized Urolithin A(UA), a natural gut-derived metabolite, as an inhibitor of Janus kinase (JAK) signaling. UA was found to broadly dampen JAK phosphorylation and the downstream signaling induced by cytokines such as type I interferons (IFN-I), type II interferons (IFN-II), and interleukin-6 (IL-6). UA can directly bind to JAK1 JH1 domain and treatment with UA attenuated autoimmune pathogenesis in Trex1-KO mice, IMQ-induced SLE and psoriasis models. Our findings unveil that UA is an anti-inflammatory metabolite that promotes immune homeostasis and could be used to treat inflammatory and autoimmune diseases.

Esophageal squamous cell carcinoma (ESCC) is still lack of clinically molecular subtyping and effective therapeutic strategies. Herein, a total of 46 paired tissue samples of esophageal squamous cell carcinoma (ESCC) were collected and subjected to a systematic proteogenomic evaluation. Consensus assessment of the ESCC-related transcriptomes and TCGA dataset revealed several consensual modes of gene expression related to ESCC specificity, with 8 plasma-detectable hub proteins that could discriminate ESCC from others. Three ESCC molecular subtypes were defined and validated based on proteome data, including pCC1 with activated immune response and best survival outcome, pCC2 as cell cycle subtype with relative worse outcome, and pCC3 with worst outcome that expressed more cell adhesion related proteins. Furthermore, we proposed potential therapeutic strategies for improving survival outcomes in patients with different ESCC molecular subtypes. This integrative proteogenomic analysis provided a novel view of ESCC-dependent molecular information.

Second generation antiandrogens, such as enzalutamide, are commonly prescribed to treat advanced prostate cancer. However, enzalutamide resistant prostate cancer (ERPC) invariably develops with more aggressive features. Management of ERPC is extremely difficult not only because available therapies cannot effectively eliminate ERPC cells but also due to enhanced growth and highly metastatic features in ERPC cells to invade distant organs. This problem is amplified by the lack of proper knowledge about suitable molecular targets in ERPC cells. Recently, we reported that Tribbles 2 (TRIB2), is overexpressed in ERPC cells and tumors and inhibition of TRIB2 kills ERPC cells via apoptosis. TRIB2 enhances cancer cell growth and invasion and confers resistance to enzalutamide, while inhibition of TRIB2 resensitizes the resistant cells. Interestingly, TRIB2 induces neuroendocrine (NE) features in ERPC cells and both the de novo and treatment-emergent NEPC cells consistently overexpress TRIB2. Though TRIB2 has emerged as a promising target, suitable inhibitors are not commercially available for clinical use. We used artificial intelligence (AI)-based molecular modeling to design a series of small chemical entities with potentially high specificity to bind with TRIB2. Extensive virtual screening and molecular editing yielded few highly selective and potent agents to inhibit TRIB2 and kill ERPC-NE/NEPC cells. One such compound (HF-125) directly binds to destabilize and degrade TRIB2 proteins involving proteasomes and is effective both in vitro and in vivo. Based on these findings, HF-125 emerges as a promising novel agent for development of a new therapeutic strategy for ERPC-NE/NEPC types of aggressive prostate cancer.

Cachexia is a devastating and multifactorial syndrome characterized by progressive loss of body weight, skeletal muscle wasting, and systemic inflammation, frequently observed in patients with advanced gastric cancer (GC) with peritoneal dissemination. Despite its clinical significance, the molecular mechanisms underlying cancer-associated cachexia remain poorly understood. In this study, comparative transcriptomic analysis using the GEMINI database identified ATP as a novel candidate cachexia-inducing factor, along with the known cachexia mediators, growth differentiation factor 11 (GDF11) and growth differentiation factor 15 (GDF15). Functional studies demonstrated that BMP7 acts as an upstream regulator that drives cachectic phenotypes by inducing the expression of GDF11 and GDF15. Knockdown of BMP7, GDF11, or GDF15 in the cachexia-inducing GC cell line, MKN45 significantly attenuated weight loss and muscle wasting in vivo. Conversely, overexpression of BMP7 in the non-cachectic GC cell line, NUGC3 induced cachexia and upregulated GDF11 and GDF15 in tumor tissues. Furthermore, clinical analysis revealed that high BMP7 expression in tumor specimens from patients with advanced GC was associated with significantly poorer overall survival. These findings identify BMP7 as a master regulator of cancer-associated cachexia through the induction of GDF11 and GDF15 and suggest its potential as a promising therapeutic target for mitigating cachexia in GC.

The recurrent outbreaks and geographical expansion of mosquito-borne arboviruses pose a significant challenge to public health worldwide. The disease outcome for arboviral infections ranges from acute febrile illness to severe conditions such as encephalitis, hemorrhagic shock, and mortality. Current treatment options for these viruses are limited to supportive care, necessitating an urgent need for a safe and effective broad-spectrum antiviral. In this study, we have identified Trifluoperazine (TFP), an FDA-approved antipsychotic, as a potent broad-spectrum antiviral against Japanese encephalitis Virus (JEV), Dengue virus (DENV) and Chikungunya virus (CHIKV) infections. The antiviral effect of TFP was also seen in the animal models of JEV and CHIKV with significantly reduced disease severity. Mechanistically, TFP treatment increased the phosphorylation of eIF2a and induced an adaptive ER stress response in diverse cell types. Alleviation of TFP-induced ER stress by chemical chaperone 4PBA abolished the antiviral activity of the drug and rescued virus replication in cells. The robust in vitro and in vivo efficacy of the drug against arboviruses highlights the potential for repurposing TFP as a broad-spectrum antiviral candidate.

The cGAS-STING pathway has been widely recognized as a critical DNA-sensing pathway that plays a broad-spectrum antiviral role. Livestock, especially pigs, represents one of the most important meat sources. In this study, we identified a key lysine 61 (K61) of porcine STING (pSTING) that plays an essential role in its degradation and antiviral signaling in a species-specific manner, with K61 as the major lysine of pSTING for K48-linked ubiquitination. After virus infection, pSTING recruits the E3 ligase, RNF5, which specifically assembles a K48-linked ubiquitin chain at K61, thereby mediating pSTING proteasomal degradation and reducing its antiviral activity. Meanwhile, the deubiquitylation of K61 is mediated mainly by deubiquitinase USP20, which enhances the stability and antiviral activity of pSTING. Together, given the relatively few lysine numbers in livestock STINGs and species-specific K61 regulation of pSTING stability and antiviral function, the K61 and its specific regulatory enzymes of pSTING could serve as potential targets for breeding of antiviral pigs and design of antiviral drugs, respectively.

Streptococcal pyrogenic exotoxin C (SpeC) is a prototypical superantigen produced by group A Streptococcus. It potently activates a broad subset of T lymphocytes via a bridging interaction involving TCR{beta}-SpeC-MHC-II. Our recent work demonstrated that SpeC induced profound release of IL-8 from human pharyngeal epithelial cells and this effect was reversible through a specific point mutation in SpeC. This study systematically investigated cellular signaling pathways using integrated transcriptomic profiling and Western blot analysis, with a focus on membrane-associated receptors and downstream intracellular signaling effectors. Our results demonstrate that this biological process is critically associated with the activation of Erk1/2, p38 MAPK and NF-{kappa}B signaling cascade. This study identifies a novel mechanism through which a bacterial superantigen target epithelial cells-the body primary physical barrier and first line of innate immune defense.

Oncolytic virotherapy is an innovative approach to cancer treatment that uses replication-competent viruses to selectively target and destroy cancer cells while leaving healthy tissues largely unaffected. Zika virus (ZIKV), a neurotropic orthoflavivirus, has recently gained attention as a potential oncolytic agent due to its ability to infect neural-derived cells and suppress tumor growth in preclinical models. Although existing studies have examined ZIKVs oncolytic effects, the mechanisms underlying these effects remain largely unexplored. Additionally, the roles of individual ZIKV proteins and their interactions with host factors remain incompletely understood. Here, we used RNA sequencing, affinity purification-mass spectrometry, and functional assays to uncover previously unidentified mechanisms underlying ZIKVs oncolytic activity in pediatric neural tumors. We found that the ZIKV non-structural proteins NS4A and NS5 exert oncolytic effects, reducing tumorsphere size. ZIKV-host protein-protein interaction networks were characterized and showed that integrin 3 (gene: ITGA3), a mediator of cell-matrix adhesion, interacts with ZIKV NS2B and NS4A. Integrin 3 was further shown to be involved in ZIKV- and NS4A-induced tumorsphere size reduction, while ITGA3 knockdown and ZIKV infection additively inhibited 3D invasion. These findings provide critical mechanistic insights that could inform the rational design of ZIKV-based virotherapies and highlight opportunities for combination treatment strategies.

Head and neck squamous cell carcinoma (HNSCC) ranks as the seventh most common cancer, with increasing incidence and mortality rates and limited therapeutic progress. The heterohexameric prefoldin complex, a highly conserved co-chaperone assembly composed of six PFDN subunits, exhibits expression levels strongly correlated with cancer progression. Among these subunits, the PFDN5 gene presents a paradoxical role in cancer biology, demonstrating both tumor-promoting and tumor-suppressive activities. Notably, the PFDN5 gene generates two distinct protein isoforms through alternative splicing, yet their individual contributions to cancer remain unexplored. In this study, we reveal that an elevated short-to-long PFDN5 alternative splice variants ratio is significantly associated with improved overall survival in HNSCC patients. Using proximity-dependent biotin identification (BioID), we mapped shared and isoform-specific protein-protein interaction networks for PFDN5. Our analysis uncovered novel proximal interactors, implicating PFDN5 isoforms in unexpected functions, including spindle organization, transcriptional complexes, and NF-{kappa}B signaling. These results provide a foundation for exploring PFDN5 isoforms as potential therapeutic targets in HNSCC.

COVID-19 has spread rapidly and caused a global pandemic making it one of the deadliest in history. Early identification of patients with coronavirus disease 2019 who may develop critical illness is of immense importance. Therefore, novel biomarkers were needed to identify patients who will suffer rapid disease progression to severe complications and death. Many treatments were adopted including the antiviral Remdesivir, the antiretroviral Lopinavir /Ritonavir and Tocilizumab. Our study aimed not only to specify high-risk factors and biomarkers of fatal outcome in hospitalized subjects with coronavirus but also to compare the efficacy of the three considered treatments to help clinicians better choose a therapeutic strategy and reduce mortality. We divided the population (n=711) into four main groups based according to the WHO ordinal severity scale. The percentage of mortality, in and out the hospital, the length of stay in the hospital, the pulmonary inflammatory lesion and its distribution, the SARS-CoV-2 IgM and IgG variations at admission, the inflammatory markers, the complete blood count, the coagulation factors and enzymes, proteins and electrolytes profile, glucose and lipid profile, and other relevant markers were measured. The significance of the observed variation was assessed by multivariate and ANOVA analyses. We succeeded to establish a novel predictive scoring model of disease progression based on a cohort of Lebanese hospitalized patients relying on the pulmonary inflammatory lesions, inflammation biomarkers such as LDH, D-Dimer, CRP, IL-6 and the lymphocyte count, the number of comorbidities and the age of the patient which all were significantly correlated with the illness severity showing best outcomes with immunomodulatory and anticoagulant treatments by the results. As top tier, Tocilizumab was more efficient than the two other treatments in non-severe cases but none of the used treatments was insanely effective alone to reduce mortality in severe cases.

Maladaptive repair of acute kidney injury (AKI) may lead to the development of chronic kidney disease (CKD) characterized by renal fibrosis. Macrophages play roles in AKI-to-CKD progression; however, the interplay between inflammation and fibrosis after AKI remains controversial and the precise role of the distinct macrophage subsets remains elusive. In the present study we identified a unique population of Trem2hi macrophages derived from the bone marrow as a mediator bridging inflammation resolution and fibrosis establishment after kidney injury. Trem2 deficient mice exhibited mitigated renal fibrosis after ischemia-reperfusion injury (IRI) while the renal injury and inflammation persisted. Mechanistically, Trem2 promoted renal inflammation resolution by facilitating macrophage efferocytosis to remove apoptotic tubule cells and reshaping the macrophage cytokine production profile. Loss of Trem2 expression led to excessive cholesterol accumulation in macrophages via Lxr-Abca1/Abcg1 axis and thus sustained pro-inflammatory cytokines production. Moreover, Trem2hi macrophages orchestrated the pro-fibrotic tubular epithelial cells and the activation of myofibroblasts through SPP1 to promote the establishment of renal fibrotic niche. Based on our findings, Trem2hi macrophages may serve as a potential therapeutic target for AKI-to-CKD in combination with anti-inflammatory remedies.

Despite of the highly potent antiretroviral therapies, HIV-1 establishes persistent infection and causes chronic inflammation in AIDS patients. Beyond CD4+ T cells, HIV-1 infects myeloid cells, including circulating monocytes and tissue-resident macrophages, and integrates with host genomes to form stable viral reservoirs. To achieve a functional HIV cure, latency-promoting agents (LPAs) have been developed for the "block-and-lock" strategy to reinforce deep HIV-1 latency and permanently silence proviruses. However, most LPAs have been tested mainly in CD4+ T cells, and their efficacy in myeloid cells remains unclear. In this study, we reported that levosimendan (LSM), a drug approved for clinic use to treat heart failures, is able to inhibit HIV lytic infection and reactivation in myeloid cells. LSM blocked viral lytic reactivation in HIV-1 latently infected monocytic cells (TH89GFP, U1) and microglial cells (HC69). LSM also inhibited HIV infection in human induced pluripotent stem cell (iPSC) derived microglia (iMG), primary human resident liver macrophages (Kupffer cells) as well as human monocyte-derived macrophages (MDMs). Furthermore, we demonstrated that overexpression of a predicted drug target of LSM, the conserved serine/threonine kinase RIOK1 (RIO kinase 1), overcomes LSMs anti-HIV effect. Overall, our studies concluded that LSM is a promising LPA to inhibit HIV-1 infection in myeloid cells in the RIOK1-dependent manner.

Colorectal cancer (CRC) poses a severe global health threat with high incidence, mortality, and poor 5-year survival rates for advanced cases despite existing treatments. This study aims to explore the role of STRIP2 in CRC progression and its underlying mechanisms. Impact of STRIP2 on CRC in vitro was investigated via CRC cell proliferation, migration, invasion, and apoptosis. The in vivo impact was investigated via nude mice models. The role of STRIP2 in CRC was investigated via transcriptomic analysis, Western blot, Co-immunoprecipitation assays and ferroptosis validations. STRIP2 is overexpressed in CRC, driving malignant phenotypes in vitro and in vivo. Mechanically, STRIP2 stabilizes the IL17 downstream effector LCN2 by blocking its K48-linked ubiquitination and degradation, enhances anti-ferroptosis of CRC cells. Oe-STRIP2 suppresses ferroptosis, boosting proliferation and reducing oxidative stress; while si-STRIP2 induces the opposite effect. This study suggests STRIP2-mediated stabilization of LCN2 and enhances CRC cells ferroptosis resistance, thus promoting CRC cell survival and mediates malignant progression in CRC, which provides a novel link between STRIP2 and ferroptosis regulation in CRC. HighlightO_LISTRIP2 is overexpressed in CRC tissues and cells C_LIO_LISTRIP2 blocks LCN2 Ubiquitination and stabilizes LCN2 C_LIO_LISTRIP2 suppresses CRC ferroptosis C_LIO_LISTRIP2 drives CRC malignant phenotypes both in vitro & in vivo C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/725308v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1baf7baorg.highwire.dtl.DTLVardef@1de15d9org.highwire.dtl.DTLVardef@16c8078org.highwire.dtl.DTLVardef@667840_HPS_FORMAT_FIGEXP M_FIG C_FIG

Inflammasomes lead to activation of inflammatory caspases, which induce pyroptosis and an inflammatory immune response to control microbial infections. Inflammasomes are tightly regulated to avoid lethal sepsis and chronic autoimmune conditions. However, posttranslational regulation of inflammatory caspases remains poorly defined. We constructed 375 individual ubiquitin ligase knockout lines by CRISPR-Cas9, performed an unbiased screening, and identified Muscle Excess 3B (MEX3B), an RNA-binding protein and ubiquitin ligase, as a positive regulator of the caspase-4 inflammasome. Genetic depletion of MEX3B inhibited not only the caspase-4 but also NLRP3 and NLRC4 inflammasomes, regarding caspase activation, pyroptosis, and secretion of inflammasome-dependent cytokines, in human cells and murine primary macrophages. This MEX3B function required its RNA-binding, but not ubiquitin ligase activity. These results suggest that MEX3B is a pan-inflammasome regulator and a potential therapeutic target for inflammation.

Human genetic studies have identified defects in multiple mechanisms that predispose the risk of developing inflammatory bowel diseases (IBD), which include alterations in adaptive and innate immune responses, epithelial integrity and regulation of the intestinal mucus layer. Despite the importance of intestinal barrier integrity in the pathogenesis of IBD, essentially all current therapies modulate the immune responses. In this study, we determined the high resolution cryo-EM structure of human NXPE1, a IBD associated protein. Based on the structural homology, we identified NXPE1 as an O-acetyltransferase. Since NXPE1 is a pseudo gene in mouse, we generated knockout mouse model that lacked two of the mouse NXPE1 homologs, Nxpe2 and Nxpe4. The O-acetylation of sialic acid on red blood cells was abolished in the double knockout mice, confirming the sialic acid O-acetyltransferase function of NXPE1 family members. These findings underscore the potential of NXPE1 as a novel therapeutic target of the intestinal barrier functions for the treatment of IBD.

Human papillomavirus 18 (HPV18) preferentially infects cervical stem cell-like cells and is strongly associated with adenocarcinoma. However, the mechanisms underlying differentiation into cervical adenocarcinoma remain unclear due to the lack of appropriate experimental models. We aimed to establish a model of HPV18-associated cervical adenocarcinoma and elucidate its molecular and cellular differentiation mechanisms. HPV18 E6/E7 were introduced into induced pluripotent stem cell-derived reserve cell-like cells (iRCs) to generate tumor models. Spatial transcriptomics and single-cell multi-omics analyses were performed to integrate histological and molecular data. A distinct component (Gland_A) exhibited morphological and immunohistochemical features of cervical adenocarcinoma and was efficiently induced in iRC-18 tumors. Gland_A showed increased chromatin accessibility and elevated expression of FOXA1, FOXA2, and ALDH1A1. Analysis of clinical samples confirmed enrichment of ALDH1A1 in HPV-associated adenocarcinomas. This model recapitulates key features of HPV18-associated cervical adenocarcinoma and provides insights into its differentiation mechanisms.

Receptor-interacting protein kinase 1 (RIPK1) is a critical regulator of programmed cell death and is implicated in various pathological conditions, particularly in mediating tumor resistance to immune checkpoint inhibitors (ICBs). In this study, we have pioneered the development of a novel cereblon (CRBN)-recruiting RIPK1 degrader, LD5095, through systematic optimization of linker and CRBN ligand portion. LD5095 demonstrates potent and selective RIPK1 degradation across cell lines, with rapid kinetics and sustained degradation over 72h post-washout. Functionally, RIPK1 degradation by LD5095 significantly sensitized Jurkat cells to TNF-induced apoptosis. Furthermore, LD5095 exhibited favorable pharmacokinetics, including metabolic stability and an extended half-life. Strikingly, in vivo, a single dose of LD5095 achieved durable RIPK1 degradation in xenograft tumors over 6 days. These findings underscore the potential of LD5095 as a chemical probe for studying RIPK1 biology and a promising candidate for cancer treatment.