Structural and thermodynamic framework for PIEZO1 modulation by small molecules.

Mechanosensitive PIEZO channels constitute potential pharmacological targets for multiple clinical conditions, spurring the search for potent chemical PIEZO modulators. Among them is Yoda1, a widely used synthetic small molecule PIEZO1 activator discovered through cell-based high-throughput screening. Yoda1 is thought to bind to PIEZO1's mechanosensory arm domain, sandwiched between two transmembrane regions near the channel pore. However, how the binding of Yoda1 to this region promotes channel activation remains elusive. Here, we first demonstrate that cross-linking PIEZO1 repeats A and B with disulfide bridges reduces the effects of Yoda1 in a redox-dependent manner, suggesting that Yoda1 acts by perturbing the contact between these repeats. Using molecular dynamics-based absolute binding free energy simulations, we next show that Yoda1 preferentially occupies a deeper, amphipathic binding site with higher affinity in PIEZO1 open state. Using Yoda1's binding poses in open and closed states, relative binding free energy simulations were conducted in the membrane environment, recapitulating structure-activity relationships of known Yoda1 analogs. Through virtual screening of an 8 million-compound library using computed fragment maps of the Yoda1 binding site, we subsequently identified two chemical scaffolds with agonist activity toward PIEZO1. This study supports a pharmacological model in which Yoda1 activates PIEZO1 by wedging repeats A and B, providing a structural and thermodynamic framework for the rational design of PIEZO1 modulators. Beyond PIEZO channels, the three orthogonal computational approaches employed here represent a promising path toward drug discovery in highly heterogeneous membrane protein systems.

Clinical evidence for efficacy of pembrolizumab in MSI-H and TMB-H advanced solid tumor: results from three cancer centers in China.

BACKGROUND: Pembrolizumab has been indicated in the treatment of solid tumors with high frequency microsatellite instability (MSI-H) or high tumor mutational burden (TMB-H); however, real-world data on the effectiveness of pembrolizumab with or without chemotherapy in this molecular subset remain limited. Our retrospective study evaluated the clinical efficacy and safety of pembrolizumab in treating advanced solid tumors with either MSI-H or TMB-H. METHODS: This retrospective study analyzed data from 116 patients with MSI-H or TMB-H advanced solid cancers who received pembrolizumab with or without chemotherapy regardless of treatment setting. We analyzed objective response rate (ORR) and progression-free survival (PFS). RESULTS: The top three cancer types were colorectal (48.6% MSI-H, 6.5% TMB-H), lung (15.4% MSI-H, 84.4% TMB-H), and gastric (15.4% MSI-H, 5.1% TMB-H). The ORR with pembrolizumab was 52.6%, including complete response (CR) observed in 8.6% (n = 10) of cases and partial responses (PR) in 43.9% (n = 51). Of the 93 patients who received first-line pembrolizumab, 52 patients achieved objective response (10 CR, 42 PR), with a median PFS of 14.0 months (95% confidence intervals [CI] 6.6-21.4). Of the 23 who received subsequent-line pembrolizumab, the ORR was 39.1%, disease control rate was 91.3%, and median PFS was 5.7 months (95% CI 3.9-7.5). Treatment-related adverse events were observed in 32 patients (27.6%), with no reported treatment-related fatal adverse events. CONCLUSION: Our study provides real-world evidence on the clinical effectiveness of pembrolizumab with or without chemotherapy in the treatment of patients with MSI-H and TMB-H advanced solid cancers.

Investigating the Properties of Triangle Terthiophene and Triphenylamine Configured Propeller-like Photochromic Dye with Ethyne Bridge.

Synthesis-oriented design led us to the construction of a propeller-like dye, containing the triangle terthiophene and triphenylamine units. It reveals typical photochromic properties with alternated UV (390 nm) and visible light (˃ 440 nm) irradiation and the dye solution (in THF) color was also toggled between yellow-green and colorless. A new absorption band was observed in visible region (415-600 nm). Additionally, the photochromic dye was highly emissive with the absolute quantum yield being 0.27. After UV light irradiation, the emission was quenched significantly (Φ = 0.08) at photo-stationary state, and thus establishing a switchable emission "on-off" system by alternated UV/visible light irradiation cycle. Detailed structural analysis was carried out based on the optimized dye structure. Both the antiparallel conformation and the distance of reactive carbon atoms (< 4.2 Å) led to the smoothly photochromic behavior.

Aristolochic acid I aggravates oxidative stress-mediated apoptosis by inhibiting APE1/Nrf2/HO-1 signaling.

Nephrotoxicity induced by aristolochic acid I (AAI) is related to redox stress and apoptosis. Apurinic/apyrimidine endonuclease 1 (APE1) has antioxidant and anti-apoptotic effects. This study investigated the potential role of APE1 in AAI-induced nephrotoxicity. Renal injury was successfully induced in C57BL/6J mice by intraperitoneal injection of AAI every other day for 28 days. Expressions of APE1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) in renal tissues of the model mice was inhibited, accompanied by oxidative damage and apoptosis. Similar results were obtained in vitro in human proximal tubular (HK-2) cells damaged by AAI. In the presence of a low concentration of the APE1 inhibitor E3330, expression of Nrf2 and HO-1 proteins in HK-2 cells was decreased and AAI-induced apoptosis was aggravated. Overexpression of APE1 in HK-2 cells promoted the expression of Nrf2 and HO-1, and alleviated apoptosis and renal injury induced by AAI. The collective findings demonstrate that AAI can inhibit the induction of oxidative stress and apoptosis by the APE1/Nrf2/HO-1 axis, leading to AAI renal injury. Targeting APE1 may be an effective therapeutic strategy to treat AA nephrotoxicity.

Photo-Induced Fluorochromism of a Star-Shaped Photochromic Dye with 2,4-Dimethylthiazole Attaching to Triangle Terthiophene.

A photochrmic triangle terthiophene dye with 2,4-dimethylthiazole attached was synthesized and shows regular photochromic properties when irradiated with UV/Vis light alternately. It was found that the attaching of 2,4-dimethylthiazole has a significant effect on both the photochromism and fluorescence of triangle terthiophene. During the photocyclizatioin prcess, not only the color but also the fluorescence of the dye in THF can be toggled between ring-open and ring-closed forms of the dye. Additionally, the absolute quantum yields (AQY) of ring-open and ring-closed forms of the dye (0.32/0.58) were greatly larger than the literature report. Along with the 254 nm light irradiation, the fluorescence color changed from deep blue (428 nm) to sky blue (486 nm) in THF. A fluorochromism cycle could be established based on the UV/visible light irradiation cycle, which provides a strategy for the design of new type fluorescent diarylethene derivatives for biological application.

Investigating the Properties of Double Triangle Terthiophene Configured Dumbbell-Like Photochromic Dye with Ethyne and 1,3-Butadiene Bridge.

Dumbbell-like photochromic dyes were constructed by incorporation of double triangle terthiophene with ethyne or 1,3-butadiene bridge. Regular photochromic behavior was investigated with alternated UV (365 nm) and Visible light (˃ 400 nm) irradiation. However, the different bridge group leads to distinct difference in their photochromic wavelength. For the ethyne bridged triangle terthiophene (DT1), the photochromic wavelength was observed around 500-700 nm (peak value: 605 nm) and the solution turned to red with 365 nm light irradiation. However, the photochromic wavelength was blue shift to 418-550 nm and the solution was turned to light yellow for 1,3-butadiene bridged dye (DT2). Both of the colored solution can be bleached via visible light irradiation. Additionally, the two dyes in THF were emissive with absolute quantum yield (QY) of 0.36/0.40. Along with the photo-induced photocyclization process, the emissive solution can be effectively quenched at photo-stationary sate (Φ = 0.05/0.04). And emission "on-off" cycle could be established based on the UV/visible light irradiation cycle.

Hippocampal asymmetry of regional development and structural covariance in preterm neonates.

Premature birth is associated with a high prevalence of neurodevelopmental impairments in surviving infants. The hippocampus is known to be critical for learning and memory, yet the putative effects of hippocampal dysfunction remain poorly understood in preterm neonates. In particular, while asymmetry of the hippocampus has been well noted both structurally and functionally, how preterm birth impairs hippocampal development and to what extent the hippocampus is asymmetrically impaired by preterm birth have not been well delineated. In this study, we compared volumetric growth and shape development in the hippocampal hemispheres and structural covariance (SC) between hippocampal vertices and cortical thickness in cerebral cortex regions between two groups. We found that premature infants had smaller volumes of the right hippocampi only. Lower thickness was observed in the hippocampal head in both hemispheres for preterm neonates compared with full-term peers, though preterm neonates exhibited an accelerated age-related change of hippocampal thickness in the left hippocampi. The SC between the left hippocampi and the limbic lobe of the premature infants was severely impaired compared with the term-born neonates. These findings suggested that the development of the hippocampus during the third trimester may be altered following early extrauterine exposure with a high degree of asymmetry.

Genetic variants in the promoters of let-7 are associated with the risk and age at onset of ischemic stroke: A case control study.

PURPOSE: Let-7 family members serve as crucial regulatory molecules in the pathogenesis of ischemic stroke. We predicted that genetic variations in the let-7 family's promoters may be linked to the risk of ischemic stroke. The connection of rs10877887 and rs13293512 in the let-7 family promoters with liability to ischemic stroke was explored in this study. PATIENTS AND METHODS: Clinical data and peripheral blood samples were collected from 914 ischemic stroke patients and 836 controls in this case-control study. All statistical analyses were carried out using SPSS. RESULTS: Our analysis results reveal that the rs10877887 TC+CC genotype in the dominant model is associated with a lower risk of ischemic stroke than the TT genotype. Individuals with heterozygous TC or homozygous CC genotypes in the male population showed higher odds of ischemic stroke than those with the wild TT genotype in rs13293512 analysis. Furthermore, there existed a multiplicative interaction between the rs10877887 C allele and the rs13293512 T allele. In the presence of the rs13293512 T allele, the effect of the rs10877887 C allele on ischemic stroke risk was increased. Similarly, in the presence of the rs10877887 C allele, the outcome of the rs13293512 T allele on ischemic stroke risk was elevated. In addition, the rs13293512 CC genotype seemed to lead to an earlier onset of ischemic stroke. CONCLUSION: Our findings indicated that these two SNPs might have a joint role in IS and could potentially act as risk markers. Detecting let-7 promoter polymorphisms could raise awareness of the risk of IS, which directed individuals with risk alleles to have regular checks at an appropriate frequency to avoid developing the disease.

Docosahexaenoic acid supplementation represses the early immune response against murine cytomegalovirus but enhances NK cell effector function.

BACKGROUND: Docosahexaenoic acid (DHA) supplementation is beneficial for several chronic diseases; however, its effect on immune regulation is still debated. Given the prevalence of cytomegalovirus (CMV) infection and because natural killer (NK) cells are a component of innate immunity critical for controlling CMV infection, the current study explored the effect of a DHA-enriched diet on susceptibility to murine (M) CMV infection and the NK cell effector response to MCMV infection. RESULTS: Male C57BL/6 mice fed a control or DHA-enriched diet for 3 weeks were infected with MCMV and sacrificed at the indicated time points postinfection. Compared with control mice, DHA-fed mice had higher liver and spleen viral loads at day 7 postinfection, but final MCMV clearance was not affected. The total numbers of NK cells and their terminal mature cell subset (KLRG1+ and Ly49H+ NK cells) were reduced compared with those in control mice at day 7 postinfection but not day 21. DHA feeding resulted in higher IFN-γ and granzyme B expression in splenic NK cells at day 7 postinfection. A mechanistic analysis showed that the splenic NK cells of DHA-fed mice had enhanced glucose uptake, increased CD71 and CD98 expression, and higher mitochondrial mass than control mice. In addition, DHA-fed mice showed reductions in the total numbers and activation levels of CD4+ and CD8+ T cells. CONCLUSIONS: These results suggest that DHA supplementation represses the early response to CMV infection but preserves NK cell effector functions by improving mitochondrial activity, which may play critical roles in subsequent MCMV clearance.

Sintilimab plus docetaxel as second-line therapy of advanced non-small cell lung cancer without targetable mutations: a phase II efficacy and biomarker study.

BACKGROUND: Single-agent immunotherapy is currently the recommended second-line therapy for patients with advanced non-small cell lung cancer (NSCLC) without targetable mutations; however, the objective response rate (ORR) remains low. This phase II study evaluated the efficacy of the combination therapy of sintilimab plus docetaxel and explored potential biomarkers for efficacy prediction. METHODS: Thirty patients with NSCLC without targetable mutations whose disease progressed from first-line platinum-based chemotherapy from October 2019 to December 2020 were enrolled in this single-arm, single-center, phase II trial. Sintilimab (200 mg) and docetaxel (75 mg/m2) were administered every 3 weeks until progression. The primary endpoint was ORR. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and safety. Biomarker analyses of blood and tissue samples were also performed. RESULTS: Among 30 patients, 11 patients had partial response, resulting in an ORR of 36.7%. The median PFS was 5.0 months (95%CI: 3.9-6.1) and OS was 13.4 months (95%CI: 5.6-21.2). The most common immune-related adverse event of any grade was hepatitis, observed in 23.3% (7/30) of patients. Treatment-emergent adverse events were manageable. Patients detected with high PD-L1 expression in circulating tumor cells (cutoff value ≥32.5% based on the median CTC-PD-L1 expression) achieved significantly higher ORR (60% versus 13.3%, p = 0.021) and significantly longer median PFS (6.0 versus 3.5 months, p = 0.011) and median OS (15.8 versus 9.0 months, p = 0.038) than those with low CTC-PD-L1 level. Patients detected with PD-L1 < 1% and CD8 ≥ 1% expression from their baseline tissue samples had significantly higher ORR (83.3% versus 12.5%, p = 0.026) but similar PFS (p = 0.62) and OS (p = 0.15). CONCLUSION: This study demonstrated the effectiveness and safety of sintilimab plus docetaxel as a second-line treatment of NSCLC without targetable mutations after progression from first-line platinum-based chemotherapy. TRIAL REGISTRATION: This study was registered in the Clinical trials registry with ClinicalTrials.gov Identifier NCT03798743 (SUCCESS).

Macrophage-derived, LRG1-enriched extracellular vesicles exacerbate aristolochic acid nephropathy in a TGFßR1-dependent manner.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by some herbal medicines, but treatment remains ineffective. We previously found that leucine-rich α-2-glycoprotein 1 (LRG1), which regulates cellular processes, plays an important role in a kidney injury model. However, the underlying mechanism by which LRG1 regulates AAN is still unknown. In this study, we established an AAN model in vivo, a coculture system of macrophages and TECs, and a macrophage/TEC conditioned media culture model in vitro. We found that macrophage infiltration promoted injury, oxidative stress, and apoptosis in TECs. Furthermore, the role of macrophages in AAN was dependent on macrophage-derived extracellular vesicles (EVs). Importantly, we found that macrophage-derived, LRG1-enriched EVs induced TEC injury and apoptosis via a TGFßR1-dependent process. This study may help design a better therapeutic strategy to treat AAN patients.

Effects of compound probiotics on intestinal barrier function and caecum microbiota composition of broilers.

Probiotics are beneficial microorganisms existing in nature and animals and can be used in livestock and poultry breeding. Here, 240 1-day-old Arbor Acre (AA) broilers were used to study the effects of compound probiotics (CP) on antioxidant capacity, intestinal barrier function and caecum microorganisms. 2‰, 3‰ or 4‰ CP were added to the basal diet. Blood, jejunum, caecum and caecum contents of broilers were collected on day 60, and the jejunum histopathological observation, oxidative stress state evaluation, intestinal barrier function mRNA level and caecum microflora composition were carried out. The results showed that CP significantly improved the growth performance of broilers in 1-30 days. Moreover, CP supplementation increased superoxide dismutase (SOD) activity, reduced malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents in serum, and increased the mRNA levels of zona occludens 1 (ZO-1), claudin-1 and occludin in the jejunum of broilers. 3‰ CP observably increased the ratio of villus height to crypt depth, and the abundance of the genus Rikenellaceae_RC9_gut_group and Phascolarctobacterium, decreased the abundance of the genus Ruminococcaceae_UCG-014, together with regulation of several genes that are responsible for signaling pathways involved in carbohydrate metabolism, amino acid metabolism and endocrine and metabolic diseases. Taken together, the supplementation of CP could reduce oxidative stress levels, increase the mRNA expression levels of tight junction (TJ)-related genes and the colonization of beneficial bacteria in the caecum, which has a promoting effect on the growth performance in broilers.

Compound probiotics alleviate cadmium-induced intestinal dysfunction and microbiota disorders in broilers.

Cadmium (Cd), a common environmental pollutant, seriously threatens the health of intestine. This research aimed to investigate the effects of compound probiotics (CP) on intestinal dysfunction and cecal microbiota dysregulation induced by Cd in broilers. A total of 240 1-day-old Arbor Acre (AA) broilers were randomly assigned to four groups. After 120 days of feeding, the jejunum tissues and cecal contents were sampled for jejunum histopathological observation, the intestinal barrier and inflammatory factors related mRNA and proteins examinations, and intestinal microbiota analysis. The results showed that Cd could cause jejunal villus damage and inflammatory cells infiltration, down-regulate the mRNA levels of intestinal barrier related genes (ZO-1, ZO-2, ZO-3, Claudin1, Claudin3, Claudin4, Occludin, and E-cadherin) and inflammatory factor related genes (IL-1ß, IL-18, IFN-γ, NF-κB), and the protein levels of Claudin1, ZO-1, Occludin, but up-regulate the Claudin2, IL-2, IL-4 and IL-10 mRNA levels. However, the addition of CP could effectively improve these changes. In addition, 16S rRNA gene sequencing analysis showed that compared with the Cd group, supplementation CP increased the abundance of Lactobacillales, Clostridiales, Firmicutes, together with regulations on the pathways responsible for energy metabolism, translation and amino acid metabolism. In conclusion, CP could improve intestinal barrier damage and intestinal microbiota disturbance induced by Cd.

Cinobufagin induces acute promyelocytic leukaemia cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the ß-catenin signalling pathway.

CONTEXT: Acute promyelocytic leukaemia (APL) is a malignant hematological tumour characterized by the presence of promyelocytic leukaemia-retinoic acid receptor A (PML-RARA) fusion protein. Cinobufagin (CBG) is one of the main effective components of toad venom with antitumor properties. However, only a few reports regarding the CBG treatment of APL are available. OBJECTIVE: We explored the effect and mechanism of action of CBG on NB4 and NB4-R1 cells. MATERIALS AND METHODS: We evaluated the viability of NB4 and NB4-R1 cells treated with 0, 20, 40, and 60 nM CBG for 12, 24, and 48 h. After treatment with CBG for 24 h, Bcl-2 associated X (Bax), B-cell lymphoma 2 (Bcl-2), ß-catenin, cyclin D1, and c-myc expression was detected using western blotting and real-time polymerase chain reaction. Caspase-3 and PML-RARA expression levels were detected using western blotting. RESULTS: CBG inhibited the viability of NB4 and NB4-R1 cells. The IC50 values of NB4 and NB4-R1 cells treated with CBG for 24 h were 45.2 nM and 37.9 nM, respectively. CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner and inhibited the ß-catenin signalling pathway. DISCUSSION AND CONCLUSION: CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the ß-catenin signalling pathway. This study proposes a novel treatment strategy for patients with APL, particularly those with ATRA-resistant APL.

Free energy and kinetics of cAMP permeation through connexin26 via applied voltage and milestoning.

The connexin family is a diverse group of highly regulated wide-pore channels permeable to biological signaling molecules. Despite the critical roles of connexins in mediating selective molecular signaling in health and disease, the basis of molecular permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3',5'-cyclophosphate (cAMP), through a connexin26 hemichannel (Cx26). First, inward and outward fluxes of cAMP molecules solvated in KCl solution were obtained from 4 µs of ± 200 mV simulations. These fluxes data yielded a single-channel permeability of cAMP and cAMP/K+ permeability ratio consistent with experimentally measured values. The results from voltage simulations were then compared with the potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP without voltage, obtained from a total of 16.5 µs of Voronoi-tessellated Markovian milestoning simulations. Both the voltage simulations and the milestoning simulations revealed two cAMP-binding sites, for which the binding constants KD and dissociation rates koff were computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under opposite voltages were derived from the milestoning PMF and revealed the interplay between voltage and channel polarity on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and nonlocal pore diameter in a position-dependent manner.

Clinical and molecular feature-based nomogram model for predicting benefit from bevacizumab combined with first-generation EGFR-tyrosine kinase inhibitor (TKI) in EGFR-mutant advanced NSCLC.

BACKGROUND: The combination of bevacizumab and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) could prolong progression-free survival (PFS) in patients with EGFR-mutant advanced non-small-cell lung cancer (NSCLC). Our study investigated the clinical and molecular factors that affect the efficacy of first-generation EGFR-TKI with or without bevacizumab and identify the subset of patients who can benefit from combination therapy. METHODS: Our study included 318 patients with EGFR-mutant locally advanced/advanced NSCLC treated with either first-generation EGFR-TKI combined with bevacizumab (A+T; n = 159) or EGFR-TKI monotherapy (T; n = 159). Two nomogram models to predict PFS and overall survival (OS), respectively, were constructed using two factors that impact EGFR-TKI efficacy: metastatic site and presence of concurrent mutations. The study cohort was stratified into 2 cohorts for training (n = 176) and validation (n = 142) of the nomogram model. Using the median score from the nomogram, the patients were stratified into two groups to analyze their survival outcome. RESULTS: The A+T group had significantly longer PFS (14.0 vs. 10.5 months; p < 0.001) and OS (37.0 vs. 26.0 months; p = 0.042) than the T group. Among the patients with concurrent mutations in tumor suppressor genes, those in the A+T group had significantly longer PFS and OS than the T group (PFS 14.5 vs. 8.0 months, p < 0.001; OS 39.0 vs. 20.0 months, p = 0.003). The higher scores from the nomograms were associated with the presence of brain/liver/pleural metastasis or concomitant gene mutations, which indicated a higher likelihood of shorter PFS and OS. The validation of the nomogram revealed that patients with lower scores had significantly longer PFS for the T group than those with higher scores (15.0 vs. 9.0 months, p = 0.002), but not for the A+T group (15.9 vs. 13.9 months, p = 0.256). CONCLUSIONS: Using a nomogram, our study demonstrated that the addition of bevacizumab may enhance the therapeutic effectiveness of EGFR-TKI by overcoming the negative impact of certain clinical and molecular factors on the efficacy of EGFR-TKI.

Targeting PRMT1-mediated FLT3 methylation disrupts maintenance of MLL-rearranged acute lymphoblastic leukemia.

Relapse remains the main cause of MLL-rearranged (MLL-r) acute lymphoblastic leukemia (ALL) treatment failure resulting from persistence of drug-resistant clones after conventional chemotherapy treatment or targeted therapy. Thus, defining mechanisms underlying MLL-r ALL maintenance is critical for developing effective therapy. PRMT1, which deposits an asymmetric dimethylarginine mark on histone/non-histone proteins, is reportedly overexpressed in various cancers. Here, we demonstrate elevated PRMT1 levels in MLL-r ALL cells and show that inhibition of PRMT1 significantly suppresses leukemic cell growth and survival. Mechanistically, we reveal that PRMT1 methylates Fms-like receptor tyrosine kinase 3 (FLT3) at arginine (R) residues 972 and 973 (R972/973), and its oncogenic function in MLL-r ALL cells is FLT3 methylation dependent. Both biochemistry and computational analysis demonstrate that R972/973 methylation could facilitate recruitment of adaptor proteins to FLT3 in a phospho-tyrosine (Y) residue 969 (Y969) dependent or independent manner. Cells expressing R972/973 methylation-deficient FLT3 exhibited more robust apoptosis and growth inhibition than did Y969 phosphorylation-deficient FLT3-transduced cells. We also show that the capacity of the type I PRMT inhibitor MS023 to inhibit leukemia cell viability parallels baseline FLT3 R972/973 methylation levels. Finally, combining FLT3 tyrosine kinase inhibitor PKC412 with MS023 treatment enhanced elimination of MLL-r ALL cells relative to PKC412 treatment alone in patient-derived mouse xenografts. These results indicate that abolishing FLT3 arginine methylation through PRMT1 inhibition represents a promising strategy to target MLL-r ALL cells.

PRMT1-mediated FLT3 arginine methylation promotes maintenance of FLT3-ITD+ acute myeloid leukemia.

The presence of FMS-like receptor tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate primitive FLT3-ITD+ leukemia cells, which are potential sources of relapse. Thus, understanding the mechanisms underlying FLT3-ITD+ AML cell persistence is essential to devise future AML therapies. Here, we show that expression of protein arginine methyltransferase 1 (PRMT1), the primary type I arginine methyltransferase, is increased significantly in AML cells relative to normal hematopoietic cells. Genome-wide analysis, coimmunoprecipitation assay, and PRMT1-knockout mouse studies indicate that PRMT1 preferentially cooperates with FLT3-ITD, contributing to AML maintenance. Genetic or pharmacological inhibition of PRMT1 markedly blocked FLT3-ITD+ AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginine 972/973, and PRMT1 promoted leukemia cell growth in an FLT3 methylation-dependent manner. Moreover, the effects of FLT3-ITD methylation in AML cells were partially due to cross talk with FLT3-ITD phosphorylation at tyrosine 969. Importantly, FLT3 methylation persisted in FLT3-ITD+ AML cells following kinase inhibition, indicating that methylation occurs independently of kinase activity. Finally, in patient-derived xenograft and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD+ AML cells relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes AML maintenance and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to eliminate FLT3-ITD+ AML cells.

Sennoside A prevents liver fibrosis by binding DNMT1 and suppressing DNMT1-mediated PTEN hypermethylation in HSC activation and proliferation.

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN) is a negative regulator of this process. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of PTEN, is selectively induced in myofibroblasts from diseased livers. Sennoside A (SA), a major purgative constituent of senna and the Chinese herb rhubarb, is widely used in China and other Asian countries as an irritant laxative. SA is reported to improve hepatic steatosis. However, the effect and mechanism of SA on liver fibrosis remain largely unknown. We recently identified a novel strategy for protecting liver fibrosis via epigenetic modification by targeting DNMT1. A Surface Plasmon Resonance (SPR) assay first reported that SA could directly bind DNMT1 and inhibit its activity. Administration of SA significantly prevented liver fibrosis, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in a CCl4 -induced mouse hepatic fibrosis model and in TGF-ß1-activated HSC-T6 cells, in vivo and in vitro. SA decreased the expression of Cyclin D1, CDK, and C-myc, indicating that SA may inhibit the activation and proliferation of TGF-ß1-induced HSC-T6. Moreover, SA significantly promoted the expression of PTEN and remarkably inhibited the expression of p-AKT and p-ERK in vitro. Blocking PTEN or overexpressing DNMT1 could reduce the effect of SA on liver fibrosis. These data suggest that SA directly binds and inhibits the activity and that attenuated DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the inhibition of the AKT and ERK pathways and prevented the development of liver fibrosis. Hence, SA might be employed as a promising natural supplement for liver fibrosis drug therapy.

Effect of CYP2D6 polymorphisms on plasma concentration and therapeutic effect of risperidone.

BACKGROUND: This study aimed to investigate the influence of CYP2D6 polymorphisms on risperidone plasma concentrations in patients with schizophrenia. Based on pharmacogenomics, we examined whether plasma concentration of risperidone is associated with clinical response and adverse side-effects. METHODS: We recruited patients with chronic schizophrenia who were then treated with risperidone. The CYP2D6 genotypes were determined using targeted sequencing. All high-frequency mutation sites of the nine exons of the gene were assayed in the present study. Plasma concentrations of risperidone and 9-hydroxyrisperidone (9-OH-RIS) were measured using high-performance liquid chromatography (HPLC). Psychiatric symptoms were monitored using The Positive and Negative Syndrome Scale (PANSS), Brief Psychiatric Rating Scale (BPRS), and Clinical Global Impression (CGI). Adverse effects were evaluated using the Barnes Akathisia Scale (BAS) and Extrapyramidal Symptom Rating Scale (ESRS). Follow-up visits were scheduled at weeks 2,4, and 8 after treatment initiation. RESULTS: Among the 76 patients, 100 C > T (rs1065852), 1038 C > T (rs1081003), 1662 G > C (rs1058164), 2851 C > T (rs16947), and 4181G > C (rs1135840) variants were detected. The most common allele was CYP2D6*10 (81.6%), whereas CYP2D6*2 (9.2%) and CYP2D6*5 (17.1%) were relatively rare. Plasma levels of risperidone and the risperidone/9-OH risperidone ratio (R/9-OH) were significantly increased in individuals with CYP2D6*10 (P < 0.05). The change in PANSS score, weight, high-density lipoprotein (HDL) level, prolactin (PRL) level, and ESRS were significantly different from baseline, between the different genotypes (P < 0.01). Moreover, individuals with CYP2D6*10 homozygous (TT) mutations were associated with higher risperidone concentration and R/9-OH ratio than those with heterozygous mutations (CT) (P < 0.01). A change from baseline in BPRS scores was observed only during week 8 and was different between heterozygous and homozygous mutations. As for the C2851T polymorphism, the incidence of adverse metabolic effects was significantly different between the C/C and C/T genotypes (P < 0.01). Regarding the G4181C polymorphisms, the changes from baseline in GLU and TG, were different between the C/C and C/G genotypes (P < 0.01). CONCLUSIONS: The genotype of CYP2D6 significantly influences the plasma concentration of risperidone and may subsequently influence the adverse side-effects following risperidone treatment, while also exerting a slight influence on clinical outcomes.