Multi-omics differences in the bone marrow between essential thrombocythemia and prefibrotic primary myelofibrosis.

Essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (pre-PMF) are Philadelphia chromosome-negative myeloproliferative neoplasms. These conditions share overlapping clinical presentations; however, their prognoses differ significantly. Current morphological diagnostic methods lack reliability in subtype differentiation, underlining the need for improved diagnostics. The aim of this study was to investigate the multi-omics alterations in bone marrow biopsies of patients with ET and pre-PMF to improve our understanding of the nuanced diagnostic characteristics of both diseases. We performed proteomic analysis with 4D direct data-independent acquisition and microbiome analysis with 2bRAD-M sequencing technology to identify differential protein and microbe levels between untreated patients with ET and pre-PMF. Laboratory and multi-omics differences were observed between ET and pre-PMF, encompassing diverse pathways, such as lipid metabolism and immune response. The pre-PMF group showed an increased neutrophil-to-lymphocyte ratio and decreased high-density lipoprotein and cholesterol levels. Protein analysis revealed significantly higher CXCR2, CXCR4, and MX1 levels in pre-PMF, while APOC3, APOA4, FABP4, C5, and CFB levels were elevated in ET, with diagnostic accuracy indicated by AUC values ranging from 0.786 to 0.881. Microbiome assessment identified increased levels of Mycobacterium, Xanthobacter, and L1I39 in pre-PMF, whereas Sphingomonas, Brevibacillus, and Pseudomonas_E were significantly decreased, with AUCs for these genera ranging from 0.833 to 0.929. Our study provides preliminary insights into the proteomic and microbiome variations in the bone marrow of patients with ET and pre-PMF, identifying specific proteins and bacterial genera that warrant further investigation as potential diagnostic indicators. These observations contribute to our evolving understanding of the multi-omics variations and possible mechanisms underlying ET and pre-PMF.

Thiophenyl Derivatives of Nicotinamide Are Metabolized by the NAD Salvage Pathway into Unnatural NAD Derivatives That Inhibit IMPDH and Are Toxic to Peripheral Nerve Cancers.

N-Pyridinylthiophene carboxamide (compound 21) displays activity against peripheral nerve sheath cancer cells and mouse xenografts by an unknown mechanism. Through medicinal chemistry, we identified a more active derivative, compound 9, and found that only analogues with structures similar to nicotinamide retained activity. Genetic screens using compound 9 found that both NAMPT and NMNAT1, enzymes in the NAD salvage pathway, are necessary for activity. Compound 9 is metabolized by NAMPT and NMNAT1 into an adenine dinucleotide (AD) derivative in a cell-free system, cultured cells, and mice, and inhibition of this metabolism blocked compound activity. AD analogues derived from compound 9 inhibit IMPDH in vitro and cause cell death by inhibiting IMPDH in cells. These findings nominate these compounds as preclinical candidates for the development of tumor-activated IMPDH inhibitors to treat neuronal cancers.

A Novel Anti-CD38 Monoclonal Antibody for Treating Immune Thrombocytopenia.

BACKGROUND: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by autoantibody-mediated platelet destruction. Treatment with CM313, a novel anti-CD38 monoclonal antibody, can result in targeted clearance of CD38-positive cells, including plasma cells. METHODS: We conducted a phase 1-2, open-label study to evaluate the safety and efficacy of CM313 in adult patients with ITP. CM313 was administered intravenously at a dose of 16 mg per kilogram of body weight every week for 8 weeks, followed by a 16-week follow-up period. The primary outcomes were adverse events and documentation of two or more consecutive platelet counts of at least 50×109 per liter within 8 weeks after the first dose of CM313. The status of peripheral-blood immune cells in patients and changes in the mononuclear phagocytic system in passive mouse models of ITP receiving anti-CD38 therapy were monitored. RESULTS: Of the 22 patients included in the study, 21 (95%) had two consecutive platelet counts of at least 50×109 per liter during the treatment period, with a median cumulative response duration of 23 weeks (interquartile range, 17 to 24). The median time to the first platelet count of at least 50×109 per liter was 1 week (range, 1 to 3). The most common adverse events that occurred during the study were infusion-related reaction (in 32% of the patients) and upper respiratory tract infection (in 32%). After CD38-targeted therapy, the percentage of CD56dimCD16+ natural killer cells, the expression of CD32b on monocytes in peripheral blood, and the number of macrophages in the spleen of the passive mouse models of ITP all decreased. CONCLUSIONS: In this study, anti-CD38 targeted therapy rapidly boosted platelet levels by inhibiting antibody-dependent cell-mediated cytotoxicity on platelets, maintained long-term efficacy by clearing plasma cells, and was associated with mainly low-grade toxic effects. (Funded by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences and others; ClinicalTrials.gov number, NCT05694767).

Efficacy and safety of human umbilical cord-derived mesenchymal stem cells in the treatment of refractory immune thrombocytopenia: a prospective, single arm, phase I trial.

Patients with refractory immune thrombocytopenia (ITP) frequently encounter substantial bleeding risks and demonstrate limited responsiveness to existing therapies. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) present a promising alternative, capitalizing on their low immunogenicity and potent immunomodulatory effects for treating diverse autoimmune disorders. This prospective phase I trial enrolled eighteen eligible patients to explore the safety and efficacy of UC-MSCs in treating refractory ITP. The research design included administering UC-MSCs at escalating doses of 0.5 × 106 cells/kg, 1.0 × 106 cells/kg, and 2.0 × 106 cells/kg weekly for four consecutive weeks across three cohorts during the dose-escalation phase, followed by a dose of 2.0 × 106 cells/kg weekly for the dose-expansion phase. Adverse events, platelet counts, and changes in peripheral blood immunity were monitored and recorded throughout the administration and follow-up period. Ultimately, 12 (with an addition of three patients in the 2.0 × 106 cells/kg group due to dose-limiting toxicity) and six patients were enrolled in the dose-escalation and dose-expansion phase, respectively. Thirteen patients (13/18, 72.2%) experienced one or more treatment emergent adverse events. Serious adverse events occurred in four patients (4/18, 22.2%), including gastrointestinal hemorrhage (2/4), profuse menstruation (1/4), and acute myocardial infarction (1/4). The response rates were 41.7% in the dose-escalation phase (5/12, two received 1.0 × 106 cells/kg per week, and three received 2.0 × 106 cells/kg per week) and 50.0% (3/6) in the dose-expansion phase. The overall response rate was 44.4% (8/18) among all enrolled patients. To sum up, UC-MSCs are effective and well tolerated in treating refractory ITP (ClinicalTrials.gov ID: NCT04014166).

Proteomics landscape and machine learning prediction of long-term response to splenectomy in primary immune thrombocytopenia.

This study aimed to identify key proteomic analytes correlated with response to splenectomy in primary immune thrombocytopenia (ITP). Thirty-four patients were retrospectively collected in the training cohort and 26 were prospectively enrolled as validation cohort. Bone marrow biopsy samples of all participants were collected prior to the splenectomy. A total of 12 modules of proteins were identified by weighted gene co-expression network analysis (WGCNA) method in the developed cohort. The tan module positively correlated with megakaryocyte counts before splenectomy (r = 0.38, p = 0.027), and time to peak platelet level after splenectomy (r = 0.47, p = 0.005). The blue module significantly correlated with response to splenectomy (r = 0.37, p = 0.0031). KEGG pathways analysis found that the PI3K-Akt signalling pathway was predominantly enriched in the tan module, while ribosomal and spliceosome pathways were enriched in the blue module. Machine learning algorithm identified the optimal combination of biomarkers from the blue module in the training cohort, and importantly, cofilin-1 (CFL1) was independently confirmed in the validation cohort. The C-index of CFL1 was >0.7 in both cohorts. Our results highlight the use of bone marrow proteomics analysis for deriving key analytes that predict the response to splenectomy, warranting further exploration of plasma proteomics in this patient population.

Metabolomics profile and machine learning prediction of treatment responses in immune thrombocytopenia: A prospective cohort study.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by antibody-mediated platelet destruction and impaired platelet production. The mechanisms underlying ITP and biomarkers predicting the response of drug treatments are elusive. We performed a metabolomic profiling of bone marrow biopsy samples collected from ITP patients admission in a prospective study of the National Longitudinal Cohort of Hematological Diseases. Machine learning algorithms were conducted to discover novel biomarkers to predict ITP patient treatment responses. From the bone marrow biopsies of 91 ITP patients, we quantified a total of 4494 metabolites, including 1456 metabolites in the positive mode and 3038 metabolites in the negative mode. Metabolic patterns varied significantly between groups of newly diagnosed and chronic ITP, with a total of 876 differential metabolites involved in 181 unique metabolic pathways. Enrichment factors and p-values revealed the top metabolically enriched pathways to be sphingolipid metabolism, the sphingolipid signalling pathway, ubiquinone and other terpenoid-quinone biosynthesis, thiamine metabolism, tryptophan metabolism and cofactors biosynthesis, the phospholipase D signalling pathway and the phosphatidylinositol signalling system. Based on patient responses to five treatment options, we screened several metabolites using the Boruta algorithm and ranked their importance using the random forest algorithm. Lipids and their metabolism, including long-chain fatty acids, oxidized lipids, glycerophospholipids, phosphatidylcholine and phosphatidylethanolamine biosynthesis, helped differentiate drug treatment responses. In conclusion, this study revealed metabolic alterations associated with ITP in bone marrow supernatants and a potential biomarker predicting the response to ITP.

Population genomic analysis reveals genetic divergence and adaptation in Brachymystax lenok.

Studying how populations in various environments differ genetically is crucial for gaining insights into the evolution of biodiversity. In order to pinpoint potential indicators of divergence and adaptation to diverse environments, we conducted a comprehensive analysis of 3,491,868 single nucleotide polymorphisms (SNPs) derived from five populations of Brachymystax lenok. We discovered significant geographic divergence among these 5 populations, which lack evidence of gene flow among them. Our results further demonstrated that the current distribution pattern of Brachymystax lenok are driven by geographical isolation and changes in oceans and rivers. We also performed genome-wide scan and identified the genes evolved to adapt the different environments, including stress response. In general, these results provide genomic support for high-level genetic divergence and the genetic basis of adaptation to different environments.

Novel orexin receptor agonists based on arene- or pyridine-fused 1,3-dihydro-2H-imidazole-2-imines.

A structurally novel class of benzo- or pyrido-fused 1,3-dihydro-2H-imidazole-2-imines was designed and evaluated in an inositol phosphate accumulation assay for Gq signaling to measure agonistic activation of the orexin receptor type 2 (OX2R). These compounds were synthesized in 4-9 steps overall from readily available starting materials. Analogs that contain a stereogenic methyl or cyclopropyl substituent at the benzylic center, and a correctly configured alkyl ether, alkoxyalkyl ether, cyanoalkyl ether, or α-hydroxyacetamido substituted homobenzylic sidechain were identified as the most potent activators of OX2R coupled Gq signaling. Our results also indicate that agonistic activity was stereospecific at both the benzylic and homobenzylic stereogenic centra. We identified methoxyethoxy-substituted pyrido-fused dihydroimidazolimine analog 63c containing a stereogenic benzylic methyl group was the most potent agonist, registering a respectable EC50 of 339 nM and a maximal response (Emax) of 96 % in this assay. In vivo pharmacokinetic analysis indicated good brain exposure for several analogs. Our combined results provide important information towards a structurally novel class of orexin receptor agonists distinct from current chemotypes.

Efficacy of systemic use of antibiotics as an adjunct to nonsurgical treatment of peri-implantitis: a meta-analysis of randomized controlled trials.

OBJECTIVES: The role of antibiotics as an adjunct to nonsurgical peri-implantitis treatment approaches has not reached a consensus. This meta-analysis aimed to review the adjunctive effect of systemic use of metronidazole and amoxicillin in patients with peri-implantitis. METHOD AND MATERIALS: PubMed, Embase, and the Cochrane Library were searched for randomized controlled trials published from inception to January 2023. RESULTS: A total of five clinical trials with a total of 211 patients were included in the analyses. No significant difference was found in the reduction of probing pocket depth at 3 and 6 months of follow-up (3 months: weighted mean difference [WMD] = -0.336, 95% CI -0.966 to 0.233, P = .231; 6 months: WMD = -0.533, 95% CI -1.654 to 0.587, P = .351). A statistically significant difference was found at 12 months of follow-up (WMD = -1.327, 95% CI -1.803 to -0.852, P < .001) between the treatment and control groups. The combined results indicated that the differences in reduction of bleeding on probing, Plaque Index score, and bone level at 6 months of follow-up were significant (P < .05). CONCLUSION: The study demonstrated that the adjunctive use of systemic metronidazole and amoxicillin did not significantly improve probing pocket depth compared to nonsurgical treatment alone, and should not be routinely recommended. However, the significant reductions in bleeding on probing, Plaque Index, and bone level at 6 months may indicate a potential effect of treating peri-implantitis with adjunctive systemic metronidazole and amoxicillin.

Multi-omics analysis of human mesenchymal stem cells shows cell aging that alters immunomodulatory activity through the downregulation of PD-L1.

Mesenchymal stem cells (MSCs) possess potent immunomodulatory activity and have been extensively investigated for their therapeutic potential in treating inflammatory disorders. However, the mechanisms underlying the immunosuppressive function of MSCs are not fully understood, hindering the development of standardized MSC-based therapies for clinical use. In this study, we profile the single-cell transcriptomes of MSCs isolated from adipose tissue (AD), bone marrow (BM), placental chorionic membrane (PM), and umbilical cord (UC). Our results demonstrate that MSCs undergo a progressive aging process and that the cellular senescence state influences their immunosuppressive activity by downregulating PD-L1 expression. Through integrated analysis of single-cell transcriptomic and proteomic data, we identify GATA2 as a regulator of MSC senescence and PD-L1 expression. Overall, our findings highlight the roles of cell aging and PD-L1 expression in modulating the immunosuppressive efficacy of MSCs and implicating perinatal MSC therapy for clinical applications in inflammatory disorders.

Machine learning analyses constructed a novel model to predict recurrent thrombosis in adults with essential thrombocythemia.

The current study involving 318 essential thrombocythemia (ET) patients with prior thrombosis was designed to identify risk factors that were predictive of recurrent thrombosis. The whole cohort was randomly split into derivation and validation cohorts. The random forest method, support vector machine with built-in recursive feature elimination model, and logistic multivariable analysis were performed in the derivation cohort, and cardiovascular risk factor (CVF) and RBC distribution width with standard deviation (RDW-SD) were finally selected as independent predictors. Subsequently we devise a 3-tiered model (low risk: 0 points; intermediate risk: 1-1.5 points; and high risk: 2.5 points) and it showed good discrimination in all cohorts. Moreover, the model was significantly correlated with rethrombosis-free survival (rTFS) (p = 0.0007 in the derivation cohort; p = 0.0019 in the validation cohort). In the whole cohort, cytoreductive therapy was more effective than antiplatelet agents alone for 10-year rTFS (p = 0.0336). No significant difference in 10-year rTFS was observed among interferon (IFN), hydroxyurea (HU), and IFN + HU therapy (p = 0.444). The present study helps identify individuals who need close monitoring and provides valuable risk signals for recurrence in ET patients with prior thrombosis.

Prediction of thrombosis in polycythemia vera: Development and validation of a multiple factor-based prognostic score system.

Background: Thrombosis is an important cause of death in patients with polycythemia vera (PV). The conventional stratification of thrombosis may ignore some potential risk factors. Objectives: This study aimed to develop and validate a multiple factor-based prediction model of thrombosis for the 2016 World Health Organization-dened PV. Methods: Clinical and next-generation sequencing data from 2 cohorts of patients with PV were analyzed. Multivariable Cox regression analyses were conducted for the identification of thrombotic risk factors and model development. Results: The study involved 372 patients in the training cohort and another 195 patients in the external validation cohort. Multivariable analyses indicated that age ≥60 years (hazard ratio [HR] 2.56, 95% CI 1.51-4.35, P < .001), cardiovascular risk factors (HR 4.22, 95% CI 2.00-8.92, P < .001), at least 1 high-risk mutation for thrombosis (mutations in DNMT3A, ASXL1, or BCOR/BCORL1) (HR 4.35, 95% CI 2.62-7.21, P < .001), and previous thrombosis (HR 5.93, 95% CI 3.29-10.68, P < .001) were independent risk factors of thrombosis. After assigning coefficient-weighted scores to each risk factor mentioned above, a multiple factor-based prognostic score system of thrombosis (MFPS-PV) was developed, classifying patients into low-risk, intermediate-risk, and high-risk groups. Patients in the 3 groups had notably different thrombosis-free survival rates (P < .001). The MFPS-PV outperformed the conventional model in discrimination power (C-statistic: 0.87 [95% CI 0.83-0.91] vs 0.80 [95% CI 0.74-0.86]). The MFPS-PV was well calibrated and remained consistent during external validation. Conclusion: The MFPS-PV, integrating genetic and clinical characteristics for the first time, shows excellent accuracy and utility for thrombosis prediction in WHO-defined PV.

[Stable Expression of Coagulation Factors by RPS6 Promoter].

OBJECTIVE: To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia. METHODS: Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene. RESULTS: The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups. CONCLUSION: After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.

Increased reactive oxygen species lead to overactivation of platelets in essential thrombocythemia.

INTRODUCTION: Platelet function, rather than platelet count, plays a crucial role in thrombosis in essential thrombocythemia (ET). However, little is known about the abnormal function of platelets in ET. Here, we investigated the functional characteristics of platelets in ET hemostasis to explore the causes of ET platelet dysfunction and new therapeutic strategies for ET. MATERIALS AND METHODS: We analyzed platelet aggregation, activation, apoptosis, and reactive oxygen species (ROS) in ET patients and JAK2V617F-positive ET-like mice. The effects of ROS on platelet function and the underlying mechanism were investigated by inhibiting ROS using N-acetylcysteine (NAC). RESULTS: Platelet aggregation, activation, apoptosis, ROS, and clot retraction were elevated in ET. No significant differences were observed between ET patients with JAK2V617F or CALR mutations. Increased ROS activated the JAK-STAT pathway, which may further influence platelet function. Inhibition of platelet ROS by NAC reduced platelet aggregation, activation, and apoptosis, and prolonged bleeding time. Furthermore, NAC treatment reduced platelet count in ET-like mice by inhibiting platelet production from megakaryocytes. CONCLUSIONS: Elevated ROS in ET platelets resulted in enhanced platelet activation, function and increased risk of thrombosis. NAC offers a potential therapeutic strategy for reducing platelet count.

Selective and brain-penetrant lanosterol synthase inhibitors target glioma stem-like cells by inducing 24(S),25-epoxycholesterol production.

Glioblastoma (GBM) is an aggressive adult brain cancer with few treatment options due in part to the challenges of identifying brain-penetrant drugs. Here, we investigated the mechanism of MM0299, a tetracyclic dicarboximide with anti-glioblastoma activity. MM0299 inhibits lanosterol synthase (LSS) and diverts sterol flux away from cholesterol into a "shunt" pathway that culminates in 24(S),25-epoxycholesterol (EPC). EPC synthesis following MM0299 treatment is both necessary and sufficient to block the growth of mouse and human glioma stem-like cells by depleting cellular cholesterol. MM0299 exhibits superior selectivity for LSS over other sterol biosynthetic enzymes. Critical for its application in the brain, we report an MM0299 derivative that is orally bioavailable, brain-penetrant, and induces the production of EPC in orthotopic GBM tumors but not normal mouse brain. These studies have implications for the development of an LSS inhibitor to treat GBM or other neurologic indications.

Molecular analysis of phenotypic heterogeneity in JAK2V617F-positive myeloproliferative neoplasms reveals a potential target for therapy.

JAK2V617F is the most frequent mutation in BCR-ABL-negative myeloproliferative neoplasms (MPNs). It is an important but not the only determinant of MPN phenotype. We performed high-throughput sequencing on JAK2V617F+ essential thrombocythaemia (ET) and polycythaemia vera (PV) patient samples to unveil factors involved in phenotypic heterogeneity and to identify novel therapeutic targets for MPN. Two concurrent mutations that may affect phenotype were identified, including mutations in SH2B3, which is primarily prevalent in PV, and SF3B1, which is more commonly mutated in ET. Next, we conducted transcriptomic analysis at the haematopoietic stem cell (HSC) and megakaryocyte (MK)-erythroid progenitor (MEP) levels. Inflammatory signalling pathways were elevated in both ET HSCs and MEPs, unlike in PV HSCs and MEPs. Notably, Wnt/ß-catenin signalling was uniquely upregulated during ET haematopoietic differentiation from HSC to MEP, and inhibiting Wnt/ß-catenin signalling blocked MK differentiation in vitro. Consistently, Wnt/ß-catenin inhibitor administration decreased platelet counts in JAK2V617F+ MPN mice by blocking MEPs and MK progenitors and by inhibiting maturation of MKs, while in wild-type mice, Wnt/ß-catenin inhibitor did not significantly reduce platelet counts. In conclusion, our findings provide new insights into the mechanisms underlying phenotypic differentiation of JAK2V617F+ PV and ET and indicate Wnt/ß-catenin signalling as a potential therapeutic target for MPN.

Highly Efficient One-Step Tagging of Endogenous Genes in Primary Cells Using CRISPR-Cas Ribonucleoproteins.

Genome editing tools have simplified the generation of knock-in gene fusions, which are widely used to study proteins in their natural context. However, strategies for tagging endogenous genes in primary cells are few and inefficient. In this study, we developed a one-step endogenous gene-tagging strategy by co-delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein complexes and chemically modified donor DNA into cells. Upon CRISPR-Cas9 blunt-end double-strand breaks, highly efficient site-specific insertion of genetic materials (3 × FLAG or eGFP) was achieved in both cell lines and primary cells. We further optimized the gene-tagging efficiency and precision by using CRISPR-Cas12a, which produces a staggered cut with a 5' overhang and thus enables precise ligation of DNA donors with a complementary 3' overhang. With high efficiency and flexibility, this platform would be extremely useful for multiplex endogenous genes tagging and further exploration of protein functions in various cell types.