MT1G, an emerging ferroptosis-related gene: A novel prognostic biomarker and indicator of immunotherapy sensitivity in prostate cancer.

BACKGROUND: Prostate cancer is a leading cause of cancer-related deaths in men worldwide. Despite advances in treatment strategies, there is still a need for novel therapeutic targets and approaches. Ferroptosis has emerged as a critical process in the development and progression of several cancers, including prostate cancer (PCA). In this study, we investigate the role of MT1G, a gene implicated in immune responses and ferroptosis, in the pathogenesis of PCA. Our objective is to elucidate its prognostic significance and its impact on the tumor microenvironment, while exploring its potential in enhancing the sensitivity to immune checkpoint inhibitor (ICI) therapy. METHODS: We utilized a combination of in silico analysis and experimental techniques to investigate the role of MT1G in PCA. First, we analyzed large-scale genomic datasets to assess the expression pattern and prognostic significance of MT1G in PCA patients. Subsequently, we performed functional assays to explore the impact of MT1G in PCA and its potential involvement in modulating immune responses. In addition, we conducted in vivo experiments to evaluate the effect of MT1G on tumor growth and response to ICI therapy. RESULTS: Our analysis revealed that MT1G expression is significantly downregulated in PCA tissues compared to normal prostate tissues and is associated with poor prognosis. Furthermore, MT1G overexpression inhibited the growth of PCA cells in vitro and in vivo. Importantly, we found that MT1G regulates the tumor microenvironment by modulating immune cell infiltration and inhibiting immunosuppressive factors. Furthermore, our study reveals a significant correlation between MT1G expression levels and the response to immune checkpoint inhibitor (ICI) therapy in prostate cancer (PCA) patients, as MT1G upregulation leads to an increase in PDL-1 expression. These findings underscore the potential of MT1G as a promising predictive biomarker for ICI therapy response in PCA patients. CONCLUSION: Our study elucidates the pivotal role played by MT1G in the pathogenesis of prostate cancer (PCA) and its profound implications for prognosis. Moreover, it raises the intriguing possibility that MT1G could pave the way for novel therapeutic approaches in PCA treatment. This potential arises from its ability to orchestrate immune infiltration within the tumor microenvironment, consequently enhancing sensitivity to immune checkpoint inhibitor (ICI) therapy. Therefore, our findings hold substantial promise for advancing our comprehension of PCA and exploring innovative therapeutic strategies.

WNKs are potassium-sensitive kinases.

With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low- or high-potassium diet. We, therefore, investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of Drosophila WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either ∼13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na+/K+-ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule Drosophila WNK activity. In vitro, potassium increased the melting temperature of Drosophila WNK, WNK1, and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of Drosophila WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, Ste20-related proline/alanine-rich kinase (SPAK). The greatest sensitivity of WNK4 to potassium occurred in the range of 80-180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of Drosophila and mammalian WNK kinases through direct effects of potassium ion on the kinase.

A Phosphorylated Intermediate in the Activation of WNK Kinases.

WNK kinases autoactivate by autophosphorylation. Crystallography of the kinase domain of WNK1 phosphorylated on the primary activating site (pWNK1) in the presence of AMP-PNP reveals a well-ordered but inactive configuration. This new pWNK1 structure features specific and unique interactions of the phosphoserine, less hydration, and smaller cavities compared with those of unphosphorylated WNK1 (uWNK1). Because WNKs are activated by osmotic stress in cells, we addressed whether the structure was influenced directly by osmotic pressure. pWNK1 crystals formed in PEG3350 were soaked in the osmolyte sucrose. Suc-WNK1 crystals maintained X-ray diffraction, but the lattice constants and pWNK1 structure changed. Differences were found in the activation loop and helix C, common switch loci in kinase activation. On the basis of these structural changes, we tested for effects on in vitro activity of two WNKs, pWNK1 and pWNK3. The osmolyte PEG400 enhanced ATPase activity. Our data suggest multistage activation of WNKs.

Identification and Characterization of a Potential Candidate Mlo Gene Conferring Susceptibility to Powdery Mildew in Rubber Tree.

Mildew resistance locus O (Mlo) gene was first found in barley as a powdery mildew susceptibility gene, and recessive mlo alleles confer durable resistance to barley powdery mildew. To identify candidate Mlo susceptibility genes in rubber tree, HbMlo12 was cloned from rubber tree clone CATAS7-33-97, which is susceptible to powdery mildew. Protein architecture analysis showed that HbMlo12 was a typical Mlo protein with seven transmembrane domains. Protein blast search in the Arabidopsis thaliana proteome database showed that HbMlo12 shared the highest similarity with AtMlo12, with 63% sequence identity. Furthermore, HbMlo12 together with the dicot powdery mildew susceptible Mlo proteins (including AtMlo2, AtMlo6, AtMlo12, tomato SlMlo1, pepper CaMlo2, pea PsMlo1, etc.) were grouped into clade V. Subcellular localization analysis in tobacco epidermal cells revealed that HbMlo12 was localized to the endoplasmic reticulum membrane. HbMlo12 was preferentially expressed in the flower and leaf of rubber tree. Moreover, its expression was significantly upregulated in response to powdery mildew inoculation. Application of exogenous ethephon caused a distinct increase in HbMlo12 expression. Additionally, HbMlo12 transcript was quickly induced by spraying salicylic acid and gibberellic acid and reached the maximum at 0.5 h after treatments. By contrast, HbMlo12 expression was downregulated by methyl jasmonate, abscisic acid, and drought stress treatments. There was no significant change in HbMlo12 expression after indole-3-acetic acid, H2O2, and wounding stimuli. Taken together, these results suggested that HbMlo12 might be a candidate Mlo gene conferring susceptibility to powdery mildew in rubber tree. The results of this study are vital in understanding Mlo gene evolution and developing new rubber tree varieties with powdery mildew resistance using reverse genetics.

Intracellular Chloride and Scaffold Protein Mo25 Cooperatively Regulate Transepithelial Ion Transport through WNK Signaling in the Malpighian Tubule.

Background With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule.Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux.ResultsIn vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, Drosophila WNK activity acutely increased. Drosophila WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux.Conclusions Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.

How activating mutations affect MEK1 regulation and function.

The MEK1 kinase directly phosphorylates ERK2, after the activation loop of MEK1 is itself phosphorylated by Raf. Studies over the past decade have revealed a large number of disease-related mutations in the MEK1 gene that lead to tumorigenesis and abnormal development. Several of these mutations result in MEK1 constitutive activity, but how they affect MEK1 regulation and function remains largely unknown. Here, we address these questions focusing on two pathogenic variants of the Phe-53 residue, which maps to the well-characterized negative regulatory region of MEK1. We found that these variants are phosphorylated by Raf faster than the wild-type enzyme, and this phosphorylation further increases their enzymatic activity. However, the maximal activities of fully phosphorylated wild-type and mutant enzymes are indistinguishable. On the basis of available structural information, we propose that the activating substitutions destabilize the inactive conformation of MEK1, resulting in its constitutive activity and making it more prone to Raf-mediated phosphorylation. Experiments in zebrafish revealed that the effects of activating variants on embryonic development reflect the joint control of the negative regulatory region and activating phosphorylation. Our results underscore the complexity of the effects of activating mutations on signaling systems, even at the level of a single protein.

Identification of a Ribose-Phosphate Pyrophosphokinase that Can Interact In Vivo with the Anaphase Promoting Complex/Cyclosome.

5-Phospho-d-ribosyl-1-diphosphate (PRPP) synthase (PRS) catalyzes the biosynthesis of PRPP, which is an important compound of metabolism in most organisms. However, no PRS genes have been cloned, let alone studied for their biological function in rubber tree. In this study, we identify a novel protein (PRS4) that interacts in vivo with rubber tree anaphase promoting complex/cyclosome (APC/C) subunit 10 (HbAPC10) by yeast two-hybrid assays. PRS4 has been cloned from rubber tree and named as HbPRS4. Blastp search in the genome of Arabidopsis thaliana showed that HbPRS4 shared the highest similarity with AtPRS4, with 80.71% identity. qRT-PCR was used to determine the expression of HbPRS4 in different tissues and under various treatments. HbPRS4 was preferentially expressed in the bark. Moreover, the expression level of HbPRS4 was significantly induced by the proteasome inhibitor MG132 treatment, suggesting it might be regulated by the ubiquitin/26S proteasome pathway. The amount of HbPRS4 transcript was obviously decreased after mechanical wounding and abscisic acid (ABA) treatments, while a slight increase was observed at 24 h after ABA treatment. HbPRS4 transcript in the latex was significantly upregulated by ethephon (ET) and methyl jasmonate (MeJA) treatments. These results suggested that HbPRS4 may be a specific substrate of HbAPC10 indirectly regulating natural rubber biosynthesis in rubber tree.

Discovery of novel TAOK2 inhibitor scaffolds from high-throughput screening.

The MAP3K (Mitogen Activated Protein Kinase Kinase Kinase) TAOK2 (Thousand-And-One Kinase 2) is an activator of p38 MAP kinase cascade that is up-regulated in response to environmental stresses. A synthetic lethal screen performed using a NSCLC (non-small cell lung cancer) cell line, and a second screen identifying potential modulators of autophagy have implicated TAOK2 as a potential cancer therapeutic target. Using a 200,000 compound high throughput screen, we identified three specific small molecule compounds that inhibit the kinase activity of TAOK2. These compounds also showed inhibition of autophagy. Based on SAR (structure-activity relationship) studies, we have predicted the modifications on the reactive groups for the three compounds.

Inhibitory Effects of Resveratrol on the Human Alveolar Rhabdomyosarcoma Cell Line PLA-802 through Inhibition of the TGF-ß1/Smad Signaling Pathway.

While anti-tumor activity of resveratrol has been demonstrated for many cancers, little is known about its effects on soft tissue sarcoma. Overexpression of TGF-ß1, a protein inhibited by resveratrol, is a well-known feature of the rhabdomyosarcoma (RMS), a type of soft tissue sarcoma. In the present study, we examined the effects of resveratrol on the human alveolar RMS (ARMS) cell line PLA-802. Cultured PLA-802 cells were treated with different concentrations of resveratrol at distinct time points and changes in their growth, cell cycle progression and TGF-ß1 signaling were assessed. MTT assay showed a decrease in the viability of PLA-802 cells treated with resveratrol (p < 0.05). Cell cycle analysis using flow cytometry revealed that resveratrol induced a significant decrease in the number of cells in S phase and an increase in the number of cells in G1 phase (p < 0.05). Furthermore, expression of TGF-ß1 and its downstream factor Smad4 mRNA and protein, assessed by RT-PCR and Western blot, were inhibited by resveratrol in a concentration- and time-dependent manner. Immunofluorescent staining results confirmed these changes in expression and showed that co-localization of TGF-ß1 with Smad4 was gradually decreased by resveratrol. Together, our results suggest that resveratrol may inhibit cell proliferation and cell cycle progression in PLA-802 cells through inhibiting activity of the TGF-ß1 signaling pathway. Our findings highlight the therapeutic potential of resveratrol for suppressing the tumorigenicity of human ARMS.

Precisely ordered phosphorylation reactions in the p38 mitogen-activated protein (MAP) kinase cascade.

The MAP kinase cascades, composed of a MAP3K, a MAP2K, and a MAPK, control switch responses to extracellular stimuli and stress in eukaryotes. The most important feature of these modules is thought to be the two double phosphorylation reactions catalyzed by MAP3Ks and MAP2Ks. We addressed whether the reactions are sequential or random in the p38 MAP kinase module. Mass spectrometry was used to track the phosphorylation of the MAP2K MEK6 by two MAP3Ks, TAO2 and ASK1, and the subsequent phosphorylation of p38α by MEK6/S*T* (where S (Ser) and T (Thr) are the two phosphorylation sites and * denotes phosphorylation). Both double phosphorylation reactions are precisely ordered. MEK6 is phosphorylated first on Thr-211 and then on Ser-207 by both MAP3Ks. This is the first demonstration of a precise reaction order for a MAP2K. p38α is phosphorylated first on Tyr-182 and then on Thr-180, the same reaction order observed previously in ERK2. Thus, intermediates were MEK6/ST* and p38α/TY*. Similarly, the phosphorylation of the p38α transcription factor substrate ATF2 occurs in a precise sequence. Progress curves for the appearance of intermediates were fit to kinetic models. The models confirmed the reaction order, revealed processivity in the phosphorylation of MEK6 by ASK1, and suggested that the order of phosphorylation is dictated by both binding and catalysis rates.

Construction and mechanistic insights of a novel ZnO functionalized rGO composite for efficient adsorption and reduction of Cr(VI).

A series of ZnO decorated reduced graphene oxide (rGO) (ZnrGOx) with different doping ratios were synthesized by the alkaline hydrothermal method using graphene oxide (GO) and Zn(NO3)2·6H2O as precursors, and subsequently used for the adsorption study of Cr(VI) in water. The morphology, crystalline phase structure, and surface elemental properties of ZnrGOx composites were revealed by XRD, SEM, BET, FT-IR, and XPS characterizations. The results showed that ZnO nanoparticles can be clearly seen on the surface of layered rGO. Meanwhile, as the doping rate increased, the C = C double bonds were broken and more carboxylic acid groups formed in ZnrGOx. In addition, the ZnrGO0.1 composite had the most excellent adsorption performance and good stability, and reusability. The adsorption removal rate of Cr(VI) can reach 99%, and the maximum adsorption amount of Cr(VI) was 68.9655 mg/g in 3 h. The isothermal and kinetic model simulations showed that Cr(VI) adsorption on ZnrGO0.1 composite is a chemical adsorption process, spontaneous and endothermic. Based on the concentrations of different valence states of Cr in the solid and liquid phases, 40% of Cr(VI) was reduced to Cr(III) on the surface of ZnrGO0.1 composite. Moreover, the adsorption-reduction mechanisms of Cr(VI) on ZnrGO0.1 composite were further elucidated. The ZnrGO0.1 composite manifested great potential as an efficient adsorbent for Cr(VI) removal.

Assessment of treatment outcomes: cytoreductive surgery compared to radiotherapy in oligometastatic prostate cancer - an in-depth quantitative evaluation and retrospective cohort analysis.

BACKGROUND: The management of oligometastatic prostate cancer, defined by its few metastatic sites, poses distinct clinical dilemmas. Debates persist regarding the most effective treatment approach, with both cytoreductive surgery and radiotherapy being key contenders. The purpose of this research is to thoroughly evaluate and compare the effectiveness of these two treatments in managing patients with oligometastatic prostate cancer. METHODS: A comprehensive search of the literature was carried out to find pertinent publications that compared the results of radiation and cytoreductive surgery for oligometastatic prostate cancer. A meta-analysis was conducted in order to evaluate both short-term and long-term survival. Furthermore, utilizing institutional patient data, a retrospective cohort research was conducted to offer practical insights into the relative performances of the two treatment regimens. RESULTS: Five relevant studies' worth of data were included for this meta-analysis, which included 1425 patients with oligometastatic prostate cancer. The outcomes showed that, in comparison to radiation, cytoreductive surgery was linked to a substantially better cancer-specific survival (CSS) [hazard ratio (HR): 0.70, 95% (CI): 0.59-0.81, P <0.001] and overall survival (OS) [HR, 0.80; 95% (CI), 0.77-0.82; P <0.01]. The two therapy groups' Progression-Free Survival (PFS) and Castration-Resistant Prostate Cancer-Free Survival (CRPCFS), however, did not differ significantly (HR: 0.56, 95% CI: 0.17-1.06; HR: 0.67, 95% CI: 0.26-1.02, respectively). Out of the 102 patients who were recruited in the retrospective cohort research, 36 had cytoreductive surgery (CRP), 36 had radiation therapy (primary lesion), and 30 had radiation therapy (metastatic lesion). The follow-up time was 46.3 months (18.6-60.0) on average. The enhanced OS in the CRP group [OS interquartile range (IQR): 45-60 months] in comparison to the radiation group (OS IQR: 39.0-59.0 months and 25.8-55.0 months, respectively) was further supported by the cohort research. Furthermore, CRP had a better OS than both radiation (primary region) and radiotherapy (metastatic region), with the latter two therapeutic methods having similar OS. CONCLUSION: This meta-analysis and retrospective research provide valuable insights into the comparative efficacy of cytoreductive surgery and radiotherapy for oligometastatic prostate cancer. While short-term survival (PFS, CRPCFS) was similar between the two groups, cytoreductive surgery exhibited superior CSS and OS. Adverse event rates were manageable in both modalities. These findings contribute to informed treatment decision-making for clinicians managing oligometastatic prostate cancer patients. Further prospective studies and randomized controlled trials are essential to corroborate these results and guide personalized therapeutic approaches for this distinct subset of patients.

Sintilimab (anti-PD-1 antibody) plus chidamide (histone deacetylase inhibitor) in relapsed or refractory extranodal natural killer T-cell lymphoma (SCENT): a phase Ib/II study.

Anti-PD-1 antibodies are a favorable treatment for relapsed or refractory extranodal natural killer T cell lymphoma (RR-ENKTL), however, the complete response (CR) rate and the duration of response (DOR) need to be improved. This phase 1b/2 study investigated the safety and efficacy of sintilimab, a fully human anti-PD-1 antibody, plus chidamide, an oral subtype-selective histone deacetylase inhibitor in 38 patients with RR-ENKTL. Expected objective response rate (ORR) of combination treatment was 80%. Patients received escalating doses of chidamide, administered concomitantly with fixed-dose sintilimab in 21-days cycles up to 12 months. No dose-limiting events were observed, RP2D of chidamide was 30 mg twice a week. Twenty-nine patients were enrolled in phase 2. In the intention-to-treat population (n = 37), overall response rate was 59.5% with a complete remission rate of 48.6%. The median DOR, progression-free survival (PFS), and overall survival (OS) were 25.3, 23.2, and 32.9 months, respectively. The most common grade 3 or higher treatment-emergent adverse events (AEs) were neutropenia (28.9%) and thrombocytopenia (10.5%), immune-related AEs were reported in 18 (47.3%) patients. Exploratory biomarker assessment suggested that a combination of dynamic plasma ctDNA and EBV-DNA played a vital prognostic role. STAT3 mutation shows an unfavorable prognosis. Although outcome of anticipate ORR was not achieved, sintilimab plus chidamide was shown to have a manageable safety profile and yielded encouraging CR rate and DOR in RR-ENKTL for the first time. It is a promising therapeutic option for this population.

The anti-inflammatory effect of kaempferol on early atherosclerosis in high cholesterol fed rabbits.

BACKGROUND: Atherosclerosis has been widely accepted as an inflammatory disease of vascular, adhesion molecules play an important role in the early progression of it. The aim of the present study was to evaluate the effect of kaempferol on the inflammatory molecules such as E-selectin (E-sel), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesionmolecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in high cholesterol induced atherosclerosis rabbit models. METHODS: Thirty male New Zealand white (NZW) rabbits were randomly divided into five groups, control group, model group, fenofibrate (12 mg/kg) group and kaempferol groups (150 mg/kg and 30 mg/kg). The rabbits were fed with a normal diet or a high cholesterol diet for 10 weeks. Levels of blood lipids, serum tumour-necrosis factor-alpha (TNF-α) and serum interleukin-1beta (IL-1ß) were detected at the end of the sixth and tenth week. Malonaldehyde (MDA) level and superoxide dismutase (SOD) activity in serum were also determined. Lesion areas of the aorta were measured with morphometry analysis after ten weeks. Gene expression of E-sel, ICAM-1, VCAM-1 and MCP-1 in aortas was determined by RT-PCR (reverse transcription-polymerase chain reaction). Immunohistochemical staining was employed to measure protein expression of E-sel, ICAM-1, VCAM-1 and MCP-1. RESULTS: Model rabbits fed with ten weeks of high-cholesterol diet developed significant progression of atherosclerosis. Compared with the control, levels of blood lipids, TNF-α, IL-1ß and MDA increased markedly in serum of model rabbits, while SOD levels decreased. Gene and protein expressions of E-sel, ICAM-1, VCAM-1 and MCP-1 in atherosclerotic aortas increased remarkably in model group. However, comparing to the model rabbits, levels of TNF-α, IL-1ß and MDA decreased significantly and serum SOD activity increased, gene and protein expressions of E-sel, ICAM-1, VCAM-1 and MCP-1 in aortas decreased significantly with the treatment of kaempferol. CONCLUSION: Kaempferol shows anti-atherosclerotic effect by modulating the gene and protein expression of inflammatory molecules.

Water and chloride as allosteric inhibitors in WNK kinase osmosensing.

Previous observations showed that chloride and osmotic stress regulate the autophosphorylation and activity of the kinase domains of WNK1 and WNK3. Further, prior crystallography on the asymmetric dimeric of the unphosphorylated WNK1 kinase domain (WNK1/S382A, WNK1/SA) revealed conserved waters in the active site. Here we show by crystallography that PEG400 applied to crystals of dimeric WNK1/SA grown in space group P1 induces de-dimerization with a change in space group to P2 1 . Both the conserved waters, referred to here as conserved water network 1 (CWN1) and the chloride binding site are disrupted by PEG400. CWN1 is surrounded and stabilized by a pan-WNK-conserved cluster of charged residues. Here we mutagenized these charges in WNK3 to probe the importance of the CWN1 to WNK regulation. Two mutations at E314 in the Activation Loop (WNK3/E314Q and WNK3/E314A) enhanced activity, consistent with the idea that the CWN1 is inhibitory. Mutations of other residues in the cluster had similar or less activity than wild-type. PEG400 activation of WNK3 was not significantly reduced in the point mutants tested. The crystallographic and assay data support a role for CWN1 and the charged cluster in stabilizing an inactive configuration of WNKs and suggest that water functions as an allosteric inhibitor of WNKs.

Identification of a Class of WNK Isoform-Specific Inhibitors Through High-Throughput Screening.

Introduction: WNK [with no lysine (K)] kinases are serine/threonine kinases associated with familial hyperkalemic hypertension (FHHt). WNKs are therapeutic targets for blood pressure regulation, stroke and several cancers including triple negative breast cancer and glioblastoma. Here, we searched for and characterized novel WNK kinase inhibitors. Methods: We used a ~210,000-compound library in a high-throughput screen, re-acquisition and assay, commercial specificity screens and crystallography to identify WNK-isoform-selective inhibitors. Results: We identified five classes of compounds that inhibit the kinase activity of WNK1: quinoline compounds, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-derived compounds. The compounds are strongly pan-WNK selective, inhibiting all four WNK isoforms. A class of quinoline compounds was identified that further shows selectivity among the WNK isoforms, being more potent toward WNK3 than WNK1. The crystal structure of the quinoline-derived SW120619 bound to the kinase domain of WNK3 reveals active site binding, and comparison to the WNK1 structure reveals the potential origin of isoform specificity. Discussion: The newly discovered classes of compounds may be starting points for generating pharmacological tools and potential drugs treating hypertension and cancer.

The emerging role of anti-PD-1 antibody-based regimens in the treatment of extranodal NK/T-cell lymphoma-associated hemophagocytic lymphohistiocytosis.

PURPOSE: Anti-PD-1 antibody (anti-PD-1 mAb) showed favorable outcomes in some patients with relapsed/refractory (r/r) extranodal NK/T-cell lymphoma (ENKTL). However, the role of anti-PD-1 antibody in NK/T-cell lymphoma-associated hemophagocytic lymphohistiocytosis (NK/T-LAHS) remains unclear. Here, we evaluated the efficacy and toxicity of anti-PD-1 antibody-based treatment in NK/T-LAHS patients. METHODS: The clinical data of 98 patients diagnosed with NK/T-LAHS at Sun Yat-sen University Cancer Center and the First Affiliated Hospital of Guangdong Pharmaceutical University from May 2014 to November 2021 were retrospectively analyzed. All patients received anti-HLH [HLH-2004 (etoposide, dexamethasone, cyclosporine A) or DEP-based (liposomal doxorubicin, etoposide, methylprednisolone)] regimen and sequential anti-ENKTL chemotherapy (ChT) combined with anti-PD-1 antibody or not. RESULTS: The overall response rate (ORR) of the anti-PD-1 mAb plus ChT regimens was higher than that of the ChT regimens (73.3% vs. 45.5%, P = 0.041). The toxicity of the anti-PD-1 mAb plus ChT regimens was tolerable. Except for higher rate of neutropenia, no significant difference in adverse events (AEs) was observed between the two groups. When the optimal response to anti-ENKTL was achieved, the median EBV DNA levels in patients who received anti-PD-1 mAb plus ChT were significantly lower than patients who received ChT only (878 copies/mL vs. 18,600 copies/mL, P = 0.001). With a median follow-up of 26.6 months (range 0-65.9 months), the median overall survival (mOS) was 3.5 months (95% CI：2.3-4.7 months). Patients treated with anti-PD-1 mAb plus ChT experienced a longer mOS than those who received ChT only [5.2 months (95% CI: 2.5-7.8 months) vs. 1.5 months (95% CI: 0.5-2.6 months), P = 0.002]. Cox multivariate analysis found that anti-PD-1 mAb was an independent prognostic factor for all NK/T-LAHS patients. CONCLUSION: In conclusion, anti-PD-1 mAb combined with ChT regimens seemed to be associated with prolonged survival in NK/T-LAHS patients and may represent a potentially promising treatment strategy for this population.

TSPAN18 facilitates bone metastasis of prostate cancer by protecting STIM1 from TRIM32-mediated ubiquitination.

BACKGROUND: Bone metastasis is a principal cause of mortality in patients with prostate cancer (PCa). Increasing evidence indicates that high expression of stromal interaction molecule 1 (STIM1)-mediated store-operated calcium entry (SOCE) significantly activates the calcium (Ca2+) signaling pathway and is involved in multiple steps of bone metastasis in PCa. However, the regulatory mechanism and target therapy of STIM1 is poorly defined. METHODS: Liquid chromatography-mass spectrometry analysis was performed to identify tetraspanin 18 (TSPAN18) as a binding protein of STIM1. Co-IP assay was carried out to explore the mechanism by which TSPAN18 inhibits STIM1 degradation. The biological function of TSPAN18 in bone metastasis of PCa was further investigated in vitro and in vivo models. RESULT: We identified that STIM1 directly interacted with TSPAN18, and TSPAN18 competitively inhibited E3 ligase tripartite motif containing 32 (TRIM32)-mediated STIM1 ubiquitination and degradation, leading to increasing STIM1 protein stability. Furthermore, TSPAN18 significantly stimulated Ca2+ influx in an STIM1-dependent manner, and then markedly accelerated PCa cells migration and invasion in vitro and bone metastasis in vivo. Clinically, overexpression of TSPAN18 was positively associated with STIM1 protein expression, bone metastasis and poor prognosis in PCa. CONCLUSION: Taken together, this work discovers a novel STIM1 regulative mechanism that TSPAN18 protects STIM1 from TRIM32-mediated ubiquitination, and enhances bone metastasis of PCa by activating the STIM1-Ca2+ signaling axis, suggesting that TSPAN18 may be an attractive therapeutic target for blocking bone metastasis in PCa.

Hydrostatic Pressure Sensing by WNK kinases.

Previous study has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell-volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N2 gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N2 gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed.

EpCAM-targeting CAR-T cell immunotherapy is safe and efficacious for epithelial tumors.

The efficacy of CAR-T cells for solid tumors is unsatisfactory. EpCAM is a biomarker of epithelial tumors, but the clinical feasibility of CAR-T therapy targeting EpCAM is lacking. Here, we report pre- and clinical investigations of EpCAM-CAR-T cells for solid tumors. We demonstrated that EpCAM-CAR-T cells costimulated by Dectin-1 exhibited robust antitumor activity without adverse effects in xenograft mouse models and EpCAM-humanized mice. Notably, in clinical trials for epithelial tumors (NCT02915445), 6 (50%) of the 12 enrolled patients experienced self-remitted grade 1/2 toxicities, 1 patient (8.3%) experienced reversible grade 3 leukopenia, and no higher-grade toxicity reported. Efficacy analysis determined two patients as partial response. Three patients showed >23 months of progression-free survival, among whom one patient experienced 2-year progress-free survival with detectable CAR-T cells 200 days after infusion. These data demonstrate the feasibility and tolerability of EpCAM-CAR-T therapy.