Identification of mechanism of the oncogenic role of FGFR1 in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most prevalent malignancy of the thyroid. Fibroblast growth factor receptor 1 (FGFR1) is highly expressed in PTC and works as an oncogenic protein in this disease. In this report, we wanted to uncover a new mechanism that drives overexpression of FGFR1 in PTC. Analysis of FGFR1 expression in clinical specimens and PTC cells revealed that FGFR1 expression was enhanced in PTC. Using siRNA/shRNA silencing experiments, we found that FGFR1 downregulation impeded PTC cell growth, invasion, and migration and promoted apoptosis in vitro, as well as suppressed tumor growth in vivo. Bioinformatic analyses predicted the potential USP7-FGFR1 interplay and the potential binding between YY1 and the FGFR1 promoter. The mechanism study found that USP7 stabilized FGFR1 protein via deubiquitination, and YY1 could promote the transcription of FGFR1. Our rescue experiments showed that FGFR1 re-expression had a counteracting effect on USP7 downregulation-imposed in vitro alterations of cell functions and in vivo suppression of xenograft growth. In conclusion, our study identifies the deubiquitinating enzyme USP7 and the oncogenic transcription factor YY1 as potent inducers of FGFR1 overexpression. Designing inhibitors targeting FGFR1 or its upstream inducers USP7 and YY1 may be foreseen as a promising strategy to control PTC development.

Remodeling tumor-associated macrophage for anti-cancer effects by rational design of irreversible inhibition of mitogen-activated protein kinase-activated protein kinase 2.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) emerges as a pivotal target in developing anti-cancer therapies. The limitations of ATP-competitive inhibitors, due to insufficient potency and selectivity, underscore the urgent need for a covalent irreversible MK2 inhibitor. Our initial analyses of The Cancer Genome Atlas database revealed MK2's overexpression across various cancer types, especially those characterized by inflammation, linking it to poor prognosis and highlighting its significance. Investigating MK2's kinase domain led to the identification of a unique cysteine residue, enabling the creation of targeted covalent inhibitors. Compound 11 was developed, demonstrating robust MK2 inhibition (IC50 = 2.3 nM) and high selectivity. It binds irreversibly to MK2, achieving prolonged signal suppression and reducing pathological inflammatory cytokines in macrophages. Furthermore, compound 11 or MK2 knockdown can inhibit the tumor-promoting macrophage M2 phenotype in vitro and in vivo. In macrophage-rich tumor model, compound 11 notably slowed growth in a dose-dependent manner. These findings support MK2 as a promising anticancer target, especially relevant in cancers fueled by inflammation or dominated by macrophages, and provide compound 11 serving as an invaluable chemical tool for exploring MK2's functions.

Design, synthesis, and biological evaluation of pyrido[2,3-d]pyrimidin-7(8H)-one derivatives as potent USP1 inhibitors.

USP1 has emerged as a novel and potential target for drug discovery in single therapeutic agents or combination with chemotherapy and molecular targeted therapy. In this study, based on the disclosed structure of ML323 and KSQ-4279, we designed and synthesized a series of pyrido[2,3-d]pyrimidin-7(8H)-one derivatives as potent USP1 inhibitors by cyclization strategy and the systematic structure-activity relationship exploration was conducted. The representative compounds 1k, 1m and 2d displayed excellent USP1/UAF inhibition and exhibited strong antiproliferation effect in NCI-H1299 cells. Further flow cytometry analysis revealed that they could arrest breast cancer cells MDA-MB-436 in the S phase. Inhibition mechanism study of compound 1m indicated these derivatives acted as reversible and noncompetitive USP1 inhibitors. Of note, the combination of compound 1m with PARP inhibitor olaparib generated enhanced cell killing in olaparib-resistant MDA-MB-436/OP cells, and compound 1m exhibited excellent oral pharmacokinetic properties in mice. Overall, our efforts may provide a reliable basis for the development of novel USP1 inhibitor as a single therapeutic agent and in combination with PARP inhibitors.

Downregulation of HNRNPA1 induced neoantigen generation via regulating alternative splicing.

BACKGROUND: Immunotherapies effectively treat human malignancies, but the low response and resistance are major obstacles. Neoantigen is an emerging target for tumor immunotherapy that can enhance anti-tumor immunity and improve immunotherapy. Aberrant alternative splicing is an important source of neoantigens. HNRNPA1, an RNA splicing factor, was found to be upregulated in the majority of tumors and play an important role in the tumor immunosuppressive microenvironment. METHODS: Whole transcriptome sequencing was performed on shHNRNPA1 SKOV3 cells and transcriptomic data of shHNRNPA1 HepG2, MCF-7M, K562, and B-LL cells were downloaded from the GEO database. Enrichment analysis was performed to elucidate the mechanisms underlying the activation of anti-tumor immunity induced by HNRNPA1 knockdown. mRNA alternative splicing was analyzed and neoantigens were predicted by JCAST v.0.3.5 and Immune epitope database. The immunogenicity of candidate neoantigens was calculated by Class I pMHC Immunogenicity and validated by the IFN-γ ELISpot assay. The effect of shHNRNPA1 on tumor growth and immune cells in vivo was evaluated by xenograft model combined with immunohistochemistry. RESULTS: HNRNPA1 was upregulated in a majority of malignancies and correlated with immunosuppressive status of the tumor immune microenvironment. Downregulation of HNRNPA1 could induce the activation of immune-related pathways and biological processes. Disruption of HNRNPA1 resulted in aberrant alternative splicing events and generation of immunogenic neoantigens. Downregulation of HNRNPA1 inhibited tumor growth and increased CD8+ T cell infiltration in vivo. CONCLUSION: Our study demonstrated that targeting HNRNPA1 could produce immunogenic neoantigens that elicit anti-tumor immunity by inducing abnormal mRNA splicing. It suggests that HNRNPA1 may be a potential target for immunotherapy.

Photosynthetic Bacteria-Hitchhiking 2D iMXene-mRNA Vaccine to Enable Photo-Immunogene Cancer Therapy.

Therapeutic mRNA vaccines have become powerful therapeutic tools for severe diseases, including infectious diseases and malignant neoplasms. mRNA vaccines encoding tumor-associated antigens provide unprecedented hope for many immunotherapies that have hit the bottleneck. However, the application of mRNA vaccines is limited because of biological instability, innate immunogenicity, and ineffective delivery in vivo. This study aims to construct a novel mRNA vaccine delivery nanosystem to successfully co-deliver a tumor-associated antigen (TAA) encoded by the Wilms' tumor 1 (WT1) mRNA. In this system, named PSB@Nb1.33C/mRNA, photosynthetic bacteria (PSB) efficiently delivers the iMXene-WT1 mRNA to the core tumor region using photo-driven and hypoxia-driven properties. The excellent photothermal therapeutic (PTT) properties of PSB and 2D iMxene (Nb1.33C) trigger tumor immunogenic cell death, which boosts the release of the WT1 mRNA. The released WT1 mRNA is translated, presenting the TAA and amplifying immune effect in vivo. The designed therapeutic strategy demonstrates an excellent ability to inhibit distant tumors and counteract postsurgical lung metastasis. Thus, this study provides an innovative and effective paradigm for tumor immunotherapy, i.e., photo-immunogene cancer therapy, and establishes an efficient delivery platform for mRNA vaccines, thereby opening a new path for the wide application of mRNA vaccines.

Umbilical cord blood-derived neutrophils possess higher viability than peripheral blood derived neutrophils.

Neutrophils, a primary type of immune cell, play critical roles in numerous biological processes. Both umbilical cord blood (UCB) and peripheral blood are rich in neutrophils. UCB is more abundant than peripheral blood, with cells generally at a more immature stage. However, comparative data between these two cell sources is lacking. This study aims to elucidate differences between UCB-derived neutrophils (UCBN) and peripheral blood-derived neutrophils (PBN). UCBN and PBN were isolated from fresh human umbilical cord blood and peripheral blood, respectively. Transcriptomic profiling was performed and compared against neutrophil RNA from three different donors. Bioinformatics analysis was employed to compare cell phenotypes. A cytokine cocktail (IFN-ß, IFN-γ, and LPS) was used to activate UCBN and PBN in vitro. A united multi-omic approach, combining transcriptomic and proteomic analysis, was followed by experimental validation through flow cytometry, cell killing assays, and proteome profiler array to verify cell functions. Transcriptomic analysis revealed that the most upregulated genes in freshly isolated umbilical cord blood neutrophils (UCBN) compared to peripheral blood neutrophils (PBN) predominantly involve neutrophil activation and cell-killing functions. Validation through flow cytometry and cell-killing experiments demonstrated that highly viable UCBN exhibited significantly stronger ovarian tumor cell-killing activity in vitro compared to PBN. Both transcriptomic and proteomic analyses indicated that the primary upregulated genes in activated UCBN are chiefly involved in biological processes related to the regulation of cytokine secretion. Integrative multi-omic analysis, including a proteome profiler array, confirmed that UCBN indeed secrete elevated levels of cytokines. In conclusion: UCBN shows higher viability and cellular activity compared with PBN, particularly in tumor cell-killing and cytokine secretion.

Cryo-EM structure of human HCN3 channel and its regulation by cAMP.

HCN channels are important for regulating heart rhythm and nerve activity and have been studied as potential drug targets for treating depression, arrhythmia, nerve pain, and epilepsy. Despite possessing unique pharmacological properties, HCN channels share common characteristics in that they are activated by hyperpolarization and modulated by cAMP and other membrane lipids. However, the mechanisms of how these ligands bind and modulate HCN channels are unclear. In this study, we solved structures of full-length human HCN3 using cryo-EM and captured two different states, including a state without any ligand bound and a state with cAMP bound. Our structures reveal the novel binding sites for cholesteryl hemisuccinate in apo state and show how cholesteryl hemisuccinate and cAMP binding cause conformational changes in different states. These findings explain how these small modulators are sensed in mammals at the molecular level. The results of our study could help to design more potent and specific compounds to influence HCN channel activity and offer new therapeutic possibilities for diseases that lack effective treatment.

Senolytic drugs dasatinib and quercetin combined with Carboplatin or Olaparib reduced the peritoneal and adipose tissue metastasis of ovarian cancer.

Chemotherapy and targeted drugs-induced senescent ovarian cancer cells that accumulate in peritoneal adipose tissue contribute significantly to chronic inflammation, disrupt homeostasis, and may fuel various aspects of cancer progression. However, the pro-senescence effects of chemotherapy and targeted drugs on adipose derived stem cells (ADSCs) within peritoneal adipose tissue remain poorly understood. In this study, we show that the first-line chemotherapy and targeted drugs can induce the cellular senescence of ADSCs in vitro and increase the aging of peritoneal adipose tissue in vivo. These treatments significantly promoted the dysregulation of glucose and lipid metabolism, including insulin resistance and liver lipid accumulation. Our study shows that dasatinib and quercetin, as senolytics, effectively restore glucose homeostasis in mice with ovarian cancer and significantly reduce adipose tissue aging. Importantly, combining these drugs with Carboplatin or Olaparib results in a marked decrease in both peritoneal and adipose tissue metastasis of ovarian cancer cells. Mechanistically, we revealed that there is crosstalk between ovarian cancer cells and senescent ADSCs. The crosstalk increases inflammatory cytokines and chemokines production in ADSCs and notably upregulates chemokine receptors on cancer cells. Collectively, these data indicate that senescent ADSCs induced by chemotherapy and targeted therapy drugs impair adipose tissue function. However, the senolytic drugs dasatinib and quercetin, can significantly ameliorate organ aging and damage induced by these treatments. Notably, dasatinib and quercetin combined with Carboplatin or Olaparib reduced the peritoneal and adipose tissue metastasis of ovarian cancer, ultimately benefiting the mice undergoing chemotherapy and targeted therapy.

Structure-based discovery of a new series of nucleoside-derived ring-opening PRMT5 inhibitors.

The ubiquitous methyltransferases employing SAM as the methyl donor have emerged as potential targets in many disease treatments, especially in anticancer. Therefore, developing SAM-competitive inhibitors of methyltransferases is of great interest to the drug research. To explore this direction, herein, we rationally designed a series of nucleoside derivatives as potent PRMT5 inhibitors with novel scaffold. The representative compounds A2 and A8 exhibited highly potent PRMT5 inhibition activity as well as good selectivity over other PRMTs and PKMTs. Further cellular experiments revealed that compounds A2 and A8 potently reduced the level of sDMA and inhibited the proliferation of Z-138 and MOLM-13 cell lines by inducing apoptosis. Moreover, compounds A8 which had favorable pharmacokinetic properties exhibited potent antitumor efficacy without the loss of body weight in a subcutaneous MOLM-13 xenograft model. In summary, our efforts provided a series of novel nucleoside analogs as potent PRMT5 inhibitors and may also offer a new strategy to develop SAM analogs as other methyltransferases' inhibitors.

Dysphagia Pattern in Early to Moderate Parkinson's Disease Caused by Abnormal Pharyngeal Kinematic Function.

Airway invasion is common in patients with Parkinson's disease (PD) and can cause serious complications. However, a PD-related dysphagic pattern has not been clearly elucidated. In this study, 53 patients with early to moderate PD were enrolled to undergo a videofluoroscopic study of swallowing evaluation (VFSS) and a battery of neuropsychological assessments. A set of VFSS variables (three visuoperceptual, nine temporal, and six spatial) were measured. The main effects of bolus viscosity and volume on airway invasion were calculated. Statistical analyses were performed to determine key kinematic factors of airway invasion for swallowing each bolus type. Airway invasion frequency was significantly higher for liquid boluses (liquid vs. pudding P < 0.001; liquid vs. honey P = 0.006). Laryngeal vestibule closure reaction time (LVCrt) was the key kinematic factor of airway invasion for 3 ml liquid swallow (P = 0.040), anterior displacement of hyoid bone was the key kinematic factor for both 5 ml and 10 ml liquid swallows (P = 0.010, 0.034, respectively). Male sex and advanced Hoehn and Yahr stage were significantly related to reduced anterior displacement of hyoid bone. These results reveal the dysphagic pattern related to PD, demonstrating that prolonged LVCrt and reduced anterior displacement of hyoid bone are two crucial kinematic factors contributing to airway invasion during the liquid swallow. In addition, hyoid bone dysfunction was correlated with disease severity and male sex. Our findings warrant further investigation of the pathophysiological mechanism of dysphagia in PD and would guide clinical intervention.

Establishing and validation of the VBV score for assessing Lung ground-glass nodules based on high-resolution computed tomography.

BACKGROUND: The widespread utilization of chest High-resolution Computed Tomography (HRCT) has prompted detection of pulmonary ground-glass nodules (GGNs) in otherwise asymptomatic individuals. We aimed to establish a simple clinical risk score model for assessing GGNs based on HRCT. METHODS: We retrospectively analyzed 574 GGNs in 574 patients undergoing HOOK-WIRE puncture and pulmonary nodule surgery from January 2014 to November 2018. Clinical characteristics and imaging features of the GGNs were assessed. We analyzed the differences between malignant and benign nodules using binary logistic regression analysis and constructed a simple risk score model, the VBV Score, for predicting the malignancy status of GGNs. Then, we validated this model via other 1200 GGNs in 1041 patients collected from three independent clinical centers in 2022. RESULTS: For the exploratory phase of this study, out of the 574 GGNs, 481 were malignant and 93 were benign. Vacuole sign, air bronchogram, and intra-nodular vessel sign were important indicators of malignancy in GGNs. Then, we derived a VBV Score = vacuole sign + air bronchogram + intra-nodular vessel sign, to predict the malignancy of GGNs, with a sensitivity, specificity, and accuracy of 95.6%, 80.6%, and 93.2%, respectively. We also validated it on other 1200 GGNs, with a sensitivity, specificity, and accuracy of 96.0%, 82.6%, and 95.0%, respectively. CONCLUSIONS: Vacuole sign, air bronchogram, and intra-nodular vessel sign were important indicators of malignancy in GGNs. VBV Score showed good sensitivity, specificity, and accuracy for differentiating benign and malignant pulmonary GGNs.

Prognostic Nomograms for Patients with Primary Sarcomatoid Carcinoma of The Urinary Bladder: Based on The SEER Database.

PURPOSE: The present study aimed to develop nomograms based on the SEER database to predict the prognosis for patients with primary sarcomatoid carcinoma of the urinary bladder (SCUB). MATERIALS AND METHODS: Patients with primary SCUB were identified in the Surveillance, Epidemiology, and End Results (SEER) database, between 1975 and 2017. Univariate and multivariable Cox analysis were conducted to identify the independent prognostic factors for developing the overall survival (OS) and cancer-specific survival (CSS) nomograms. Then, concordance index (C-index), receiver operating characteristic (ROC) curve and calibration curve were used to evaluate the accuracy of the nomogram model. In addition, the model was further compared with TNM staging system. RESULTS: A total of 238 eligible patients with primary SCUB were selected from the SEER database. As suggested by Cox-analysis, age, sex, T stage, M stage, tumor size, and surgery type of primary site were identified as the independent factors for predicting both OS and CSS. We developed OS and CSS nomograms with a favorable C-index by using these prognostic factors. The C-indexes of the OS and CSS nomogram in the present study were 0.738 (0.701-0.775) and 0.763 (0.724-0.802), which were superior to those of the AJCC TNM staging with 0.621 (0.576-0.666) and 0.637 (0.588-0.686) respectively, showing better discriminatory ability. Subsequently, the ROC curves showed that the 1-, 3- and 5-year AUCs (area under the curve) of OS nomogram (i.e., 0.793, 0.807 and 0.793) were higher than those of the TNM stage((i.e., 0.659, 0.676, 0.659). Similarly, as for CSS model, them ((i.e., 0.823, 0.804 and 0.804) were aslo exceed those of TNM stage((i.e., 0.683, 0.682, 0.682). Furthermore, the calibration curves indicated a good consistency between the predictive survival and the actual survival. Finally, patients were stratified by risk, and Kaplan-Meier survival curve suggested that the prognosis of the low-risk group was significantly better than that of the high-risk group. CONCLUSION: We developed nomograms with the SEER database, which could help predict the prognosis of SCUB individuals more accurately.

Screening and optimization of phage display cyclic peptides against the WDR5 WBM site.

Of the various WD40 family proteins, WDR5 is a particularly important multifunctional adaptor protein that can bind to several protein complexes to regulate gene activation, so it was considered as a promising epigenetic target in anti-cancer drug development. Despite many inhibitors having been discovered directing against the arginine-binding cavity in WDR5 called the WIN site, the side hydrophobic cavity called the WBM site receives rather scant attention. Herein, we aim to obtain novel WBM-targeted peptidic inhibitors with high potency and selectivity. We employed two improved biopanning approaches with a disulfide-constrained cyclic peptide phage library containing 7 randomized residues and identified several peptides with micromole binding activity by docking and binding assay. To further optimize the stability and activity, 9 thiol-reactive chemical linkers were utilized in the cyclization of the candidate peptide DH226027, which had good binding affinity. This study provides an effective method to discover potent peptides targeting protein-protein interactions and highlights a broader perspective of peptide-mimic drugs.

Design and synthesis of 1H-pyrazolo[3,4-d]pyrimidine derivatives as hematopoietic progenitor kinase 1 (HPK1) inhibitors.

Despite immune checkpoint inhibitors' tremendous success in the treatment of tumors, the moderate response rate limits their widespread use. Hematopoietic progenitor kinase 1 (HPK1) is served as an essential negative regulator of T-cell receptor, which has been identified as a promising target for enhancing antitumor immunity. However, the development of a selective HPK1 inhibitor is still challenging. Herein, we reported a novel series of 1H-pyrazolo[3,4-d]pyrimidine derivatives as HPK1 inhibitors by structure-based rational design. The optimal compound 10n significantly inhibited HPK1 with an IC50 value of 29.0 nM and the phosphorylation of SLP76 at a concentration as low as 0.1 µM. Furthermore, compound 10n exhibited good selectivity over a panel of 25 kinases, including GLK from the same MAP4K family. Together, the current study provided a novel, potent, and selective HPK1 inhibitor, acting as a lead compound for the future development of cancer immunotherapy.

Structure-based discovery of potent USP28 inhibitors derived from Vismodegib.

Ubiquitin-specific proteases (USPs) 28 is overexpressed in multiple types of cancers. The development of potent USP28 inhibitors is still in primitive stage. We previously reported our discovery of Vismodegib as a USP28 inhibitor by screening a commercially available drug library. Herein, we report our efforts to solve the cocrystal structure of Vismodegib bound to USP28 for the first time and subsequent structure-based optimization leading to a series of Vismodegib derivatives as potent USP28 inhibitors. Based on the cocrystal structure, elaborative SARs exploration was carried out to afford much more potent USP28 inhibitors than Vismodegib. The representative compounds 9l, 9o and 9p bearing high potency on USP28 showed high selectivity over USP2, USP7, USP8, USP9x, UCHL3 and UCHL5. The detailed cellular assay suggested that compounds 9l, 9o and 9p could cause cytotoxicity in both human colorectal cancer and lung squamous carcinoma cells and significantly enhance the sensitivity of colorectal cancer cells to Regorafenib. Further immunoblotting analysis indicated that compounds 9l, 9o and 9p could dose-dependently down-regulate the cellular level of c-Myc through ubiquitin-proteasome system and anti-cancer effects could mainly be attributed to their inhibition on USP28 but not involving the Hedgehog-Smoothened pathway. Thus, our work provided a series of novel and potent USP28 inhibitors derived from Vismodegib and may contribute to the development of USP28 inhibitors.

Localized Alkaline Environment via In Situ Electrostatic Confinement for Enhanced CO2-to-Ethylene Conversion in Neutral Medium.

Electrocatalytic CO2 reduction reaction (CO2RR) is one of the most promising routes to facilitate carbon neutrality. An alkaline electrolyte is typically needed to promote the production of valuable multi-carbon molecules (such as ethylene). However, the reaction between CO2 and OH- consumes a significant quantity of CO2/alkali and causes the rapid decay of CO2RR selectivity and stability. Here, we design a catalyst-electrolyte interface with an effective electrostatic confinement of in situ generated OH- to improve ethylene electrosynthesis from CO2 in neutral medium. In situ Raman measurements indicate the direct correlation between ethylene selectivity and the intensities of surface Cu-CO and Cu-OH species, suggesting the promoted C-C coupling with the surface enrichment of OH-. Thus, we report a CO2-to-ethylene Faradaic efficiency (FE) of 70% and a partial current density of 350 mA cm-2 at -0.89 V vs the reversible hydrogen electrode. Furthermore, the system demonstrated a 50 h stable operation at 300 mA cm-2 with an average ethylene FE of ∼68%. This study offers a universal strategy to tune the reaction micro-environment, and a significantly improved ethylene FE of 64.5% was obtained even in acidic electrolytes (pH = 2).

Development of a series of quinazoline-2,5-diamine derivatives as potent hematopoietic progenitor kinase 1 (HPK1) inhibitors.

Hematopoietic progenitor kinase 1 (HPK1) is a serine/threonine kinase that serves as the negative regulator of multiple immune signaling pathways. Genetic studies using HPK1 knockout and kinase-dead mice suggested that inhibiting HPK1 either alone or in combination with immune checkpoint blockade could be a promising strategy in cancer immunotherapy. Herein, we report the design, synthesis and structure-activity relationship (SAR) study of a series of potent HPK1 inhibitors bearing quinazoline-2,5-diamine scaffold. Three rounds of SAR exploration led to the identification of 9h, the most potent compound in this series which harbors a 2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl substituent. Further biological assessments using human immune cells demonstrated that 9h could strongly inhibit downstream phosphorylation, augment interleukin-2 (IL-2) production and reverse prostaglandin E2 (PGE2)-induced immune suppression. Overall, our study on these quinazoline-2,5-diamine derivatives provided not only a tool compound for the community to help with elucidating the HPK1 pharmacology, but also a reliable reference for subsequent development of HPK1 inhibitors.