ObjectiveIntrahepatic cholestasis of pregnancy (ICP) is a liver disorder with limited treatment options. This study investigated the therapeutic potential of resveratrol (RES) and its underlying mechanisms, focusing on the gut-liver axis and bile acid metabolism in an estrogen-induced ICP rat model.MethodsPregnant rats were randomized into Sham, ICP (induced by 17β-estradiol), and ICP+RES (15, 30, 60 mg/kg) groups. Systemic and hepatic inflammation, liver function, histopathology, and intestinal barrier integrity were assessed. Hepatic bile acid profiles were analyzed by UHPLC-MS/MS, and gut microbiota was evaluated by 16S rRNA sequencing. The role of gut microbiota was further examined via fecal microbiota transplantation (FMT) in pseudogerm-free rats. Key proteins in the FXR signaling pathway were analyzed by Western blotting.ResultsRES treatment dose-dependently alleviated ICP manifestations, including reducing serum levels of total bile acids, total bilirubin, and liver enzymes (AST, ALT, ALP), while mitigating systemic and hepatic inflammation. It also restored intestinal barrier integrity and corrected gut microbiota dysbiosis. FMT from RES-treated donors recapitulated these therapeutic effects in recipient ICP rats. Furthermore, RES reversed the hepatic bile acid imbalance by reducing primary bile acids and increasing beneficial secondary bile acids. Mechanistically, RES upregulated the expression of FXR and its downstream targets, including SHP, BSEP, UGT2B4, and CYP1A1.ConclusionRES effectively ameliorated ICP through multi-faceted mechanisms involving the attenuation of inflammation, restoration of gut microbiota and intestinal barrier, and correction of bile acid homeostasis via activation of the FXR signaling pathway. Our findings highlight RES as a promising multi-mechanistic therapeutic candidate for ICP.

Background:Uterine leiomyosarcoma (ULMS) is a rare, aggressive uterine malignancy with high misdiagnosis rates, poor prognosis, and limited molecular biomarkers. Its pathogenesis, links between specific genes and the tumor immune microenvironment (TIME), and applications of machine learning (ML) and Mendelian randomization (MR) remain understudied. Methods:Multi-cohort data (4 GEO datasets, TCGA-SARC, single-cell sequencing) were integrated. Differentially expressed genes (DEGs) and WGCNA-derived key modules identified "InteGenes". 113 ML algorithms were compared to build a diagnostic model (top: GBM, core genes = "Mgenes"). CIBERSORT analyzed TIME; MR explored Mgenes-ULMS causal links. Results:96 InteGenes enriched in cell cycle/p53/DNA repair pathways. The GBM model had training AUC = 1 and validation accuracy 92.3-100%; 36 Mgenes (e.g., TRIP13, AUC = 0.972) showed diagnostic value. Mgenes correlated with TIME (upregulated Mgenes ↔ M2 TAMs/Tregs; downregulated ↔ effector cells). MR found no genetic causality between Mgenes and ULMS. Conclusion:InteGenes reflect ULMS pathogenesis; the GBM model and Mgenes are promising diagnostic tools. Mgenes modulate ULMS's TIME, offering immunotherapeutic targets. This study advances ULMS molecular/immune understanding for translational research.

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease with limited treatment options. The COMMODORE 2 study demonstrated that the novel C5 inhibitor crovalimab had comparable safety and non-inferior efficacy to eculizumab. We evaluated the safety and efficacy of crovalimab compared with eculizumab in the Chinese subpopulation of the global COMMODORE 2 study. Adult patients with PNH without previous complement inhibitor therapy were randomized (2:1) to crovalimab or eculizumab for 24 weeks. The co-primary endpoints were the proportions of patients achieving hemolysis control from Weeks 5–25 and those with transfusion avoidance from baseline to Week 25. The secondary endpoints included the proportion of patients with breakthrough hemolysis and hemoglobin stabilization from baseline to Week 25 and the mean change from baseline in FACIT-Fatigue score at Week 25. Safety data were assessed from baseline to the clinical cut-off (16 November 2022). The demographic and baseline characteristics of the 81 patients included (eculizumab: 27; crovalimab: 54) were well-balanced. In the crovalimab and eculizumab arms, respectively, hemolysis control was achieved in 88.3% and 86.8%, transfusion avoidance in 72.2% and 74.1%, hemoglobin stabilization in 72.2% and 70.4%, and breakthrough hemolysis in 5.6% and 11.1%. The FACIT-Fatigue score improvement was greater with crovalimab. Adverse events (81.5% vs. 88.9%) and grade 3–5 adverse events (24.1% vs. 33.3%) were less frequent with crovalimab. Over 24 weeks, 91% of patients achieved complete terminal complement inhibition with crovalimab. Crovalimab demonstrated at least comparable treatment performance and acceptable safety in Chinese patients with PNH. Clinical trial registration ClinicalTrials.gov identifier NCT04434092 (study registration was first posted on 16th June 2020).

Preeclampsia (PE) is a severe pregnancy-specific hypertensive disorder characterized by immune microenvironment dysregulation at the maternal-fetal interface, with decidual macrophage phenotypic imbalance being a key pathological feature. The Ghrelin/growth hormone secretagogue receptor-1a (GHSR-1a) axis exerts immunomodulatory and anti-inflammatory effects, but its role in regulating decidual macrophage infiltration and phenotypic marker expression in PE remains unclear. In this study, we first detected the expression of the Ghrelin/GHSR-1a axis in decidual tissues from 10 healthy pregnant women and 12 PE patients via immunohistochemistry (IHC). We then established a lipopolysaccharide (LPS)-induced PE-like rat model to investigate the axis's functional role and underlying mechanisms. Intriguingly, clinical analysis revealed a severity-dependent compensatory escalation of the Ghrelin/GHSR-1a axis in PE decidual tissues, potentially representing an endogenous antagonistic response to pregnancy-associated pathological stress. In the animal model, exogenous Ghrelin supplementation reversed LPS-induced PE-like phenotypes, including hypertension, proteinuria, fetal growth restriction (FGR), and placental dysfunction, and alleviated pathological damage to the maternal liver, kidney, and placenta. Mechanistically, Ghrelin modulated decidual macrophage phenotypic marker expression by downregulating the M1 marker CD86 and upregulating the M2 marker CD163 and promoted trophoblast invasion and spiral artery remodeling by restoring laminin, α-cytokeratin 7 (α-CK7), and α-smooth muscle actin (α-SMA) expression in placental tissue. All protective effects of Ghrelin were abrogated by co-administration of D-lys-3-GHRP-6, a specific GHSR-1a antagonist, confirming the dependence on the Ghrelin/GHSR-1a axis. Collectively, our findings suggest that the Ghrelin/GHSR-1a axis is compensatorily upregulated in PE and may exert a protective role by regulating decidual macrophage phenotypic marker expression and improving placental function, providing preliminary evidence that this axis merits further investigation as a potential research target for PE.

Ovarian cancer (OC) has a dismal prognosis due to late diagnosis and platinum resistance, with the molecular mechanisms linking cuproptosis, lncRNAs, and the tumor microenvironment (TME) remaining poorly defined. LncRNA ENSG00000253374 was previously identified as a prognostic biomarker for OC, while its correlation with cuproptosis and platinum resistance remains unclear. Integrated bulk RNA-seq (TCGA-OV) and single-cell RNA-seq (GSE300897) analyses were performed, combined with in vitro CCK-8 assays, intracellular Cu⁺ detection, survival analysis, WGCNA, pseudotime trajectory, CellChat, virtual knockout, and functional enrichment analyses. OC cells with ENSG00000253374 knockdown were subjected to cuproptosis induction, and GLS-centered co-expression patterns and TME features were characterized. ENSG00000253374 knockdown correlates with altered cellular response to cuproptosis induction, and shows close expression correlation with GLS. The ENSG00000253374–GLS correlated signature is linked to abnormal glutamine metabolism, cell cycle and stemness features, and is associated with immunosuppressive myeloid differentiation and enhanced pro-resistance intercellular communication that correlate with platinum refractoriness. WGCNA constructed a GLS-centered co-expression network enriched in immune and stromal remodeling pathways; in silico GLS perturbation predicted altered stemness, angiogenesis and apoptotic signaling signatures. Elevated GLS expression in tumor and stromal cells coincides with stronger TME crosstalk and dominant M2 macrophage populations. LncRNA transcripts of the ENSG00000253374 locus are associated with platinum-resistant ovarian cancer phenotypes via a GLS-related correlative signature, alongside disrupted cuproptosis and metabolic-immune remodeling. Our in vitro assays reflect overall transcriptional activity of this genomic locus rather than single splice variant function. ENSG00000253374 and GLS may serve as candidate prognostic biomarkers. Combinatorial interventions targeting this correlative axis together with cuproptosis inducers or immune modulators could provide potential strategies to relieve platinum resistance in OC.

Objective:To analyze the risk factors associated with gastrointestinal bleeding in pediatric patients with Henoch-Schönlein purpura (HSP), with the goal of enhancing early diagnosis and treatment, preventing complications, and improving patient outcomes. Methods:The study involved 23 pediatric patients with HSP who experienced gastrointestinal bleeding, forming the study group. They were admitted to our hospital from June 2023 to June 2024. For comparison, a control group composed of 44 children with HSP but without gastrointestinal bleeding, admitted during the same timeframe, was established. Data on clinical characteristics, laboratory results, and imaging findings were collected. Both univariate and multivariate analyses were conducted to identify potential risk factors. Results:A total of 67 children with HSP were divided into two groups: those with gastrointestinal bleeding (23 cases) and those without (44 cases). The occurrence of abdominal pain and incidence of intestinal wall thickening detected by gastrointestinal ultrasonography were significantly higher in the group with gastrointestinal bleeding compared to the group without bleeding (P < 0.05). Univariate analysis revealed significant differences in eight laboratory parameters (WBC, NE, NLR, PCT, CRP, D-dimer, Fib, TT), all statistically significant (P < 0.05). Multivariate analysis identified three independent risk factors for gastrointestinal bleeding in children with HSP: abdominal pain (OR = 2.334, 95% CI: 0.458-11.886, P = 0.010), a PCT level above 0.12 ng/ml (OR = 10.010, 95% CI: 1.208-82.929, P = 0.033), and a D-dimer level exceeding 1.87 mg/L (OR = 3.407, 95% CI: 1.022-17.473, P < 0.001). Conclusion:The findings confirmed that abdominal pain, elevated PCT levels, and increased D-dimer are significant independent risk factors for gastrointestinal bleeding in pediatric patients with Henoch-Schönlein purpura. Clinicians should monitor these indicators attentively to enhance patient management and outcomes.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder characterized by complement-mediated hemolysis, thrombosis, and bone marrow failure. Iptacopan, an oral factor B inhibitor, has demonstrated efficacy in managing PNH but has not been studied in patients with severe renal insufficiency. We report a case of a PNH patient with end-stage renal disease who required renal replacement therapy and had a peritoneal dialysis catheter placed during treatment. After switching from eculizumab to iptacopan, the patient achieved transfusion independence, sustained hematologic improvement, and resolution of both intravascular and extravascular hemolysis. Iptacopan was well tolerated, with only mild adverse effects and no breakthrough hemolysis or infections. This case highlights the potential of iptacopan as a therapeutic option in PNH patients with severe renal impairment requiring dialysis.

Metabolic and bariatric surgery (MBS) can effectively improve obesity and its related complications, but it may lead to side effects such as iron deficiency (ID) and iron deficiency anemia (IDA). However, the specific risk factors for ID and IDA after laparoscopic sleeve gastrectomy (LSG) remain unclear. We retrospectively enrolled 465 patients who underwent LSG from April 2014 to December 2021. The risk factors for postoperative ID and IDA were evaluated using logistic regression analysis. Patients who underwent LSG from December 2020 to December 2021 were given low-dose iron and vitamin B12 orally based on the iron-containing multivitamin supplementation throughout the year after surgery. We analyzed the incidence of postoperative ID and IDA in patients who received additional prophylactic treatment compared with those who did not. One hundred one (21.7

BACKGROUND:Spleen tyrosine kinase inhibitors are potential treatment options for warm autoimmune haemolytic anaemia. This study aimed to assess the preliminary efficacy and safety of sovleplenib-an oral spleen tyrosine kinase inhibitor-in patients with warm autoimmune haemolytic anaemia in China. Here we report on the phase 2 results. METHODS:This randomised, double-blind, placebo-controlled, phase 2 part from the phase 2/3 study was conducted at 13 centres in China. Eligible patients, aged 18-75 years, with an Eastern Cooperative Oncology Group (ECOG) performance status of no more than 2, had primary or secondary warm autoimmune haemolytic anaemia (stable underlying disease not requiring drug intervention) with no response to previous glucocorticoid treatment, haemoglobin of less than 100 g/L with active haemolysis, and a positive direct antiglobulin test. The study comprised two periods; patients were randomly assigned (3:1) to receive sovleplenib or placebo at 300 mg orally once a day in the 8-week double-blind period. Upon completion, all patients entered an open-label treatment period for at least 16 weeks and received sovleplenib 300 mg once a day until 24 weeks after the last patient was randomly assigned. The primary endpoint for phase 2 of the trial was overall haemoglobin response rate (haemoglobin ≥100 g/L with an increase of ≥20 g/L from baseline at least once, and haemoglobin not affected by rescue therapy, such as red blood cell transfusions, intravenous immunoglobulin, and glucocorticoids) by week 24. Efficacy analyses in the 0-8 week double-blind period included all patients who were randomly assigned, analysed by intention-to-treat. Safety analysis in the double-blind period included patients in the intention-to-treat population who received at least one dose of the study medication. This phase 2/3 study is registered with ClinicalTrials.gov, NCT05535933, and the phase 3 part is ongoing. FINDINGS:Between Sept 26, 2022, and May 9, 2023, 34 patients were screened and 21 patients (four [19%] male and 17 [81%] female) were enrolled in the study and randomly assigned to receive either sovleplenib (n=16) or placebo (n=5). All 21 patients completed the 0-8-week double-blind treatment and entered the open-label treatment period. The overall haemoglobin response rate was 67% (14 of 21 patients) by week 24, and durable haemoglobin response rate was 48% (ten of 21 patients) by week 24. During the 0-8-week double-blind treatment, 13 (81%) of 16 patients in the sovleplenib group versus five (100%) of five patients taking placebo reported treatment-emergent adverse events (TEAEs), and four (25%) of 16 patients versus four (80%) of five patients reported grade 3 adverse events. Although all 21 patients had a TEAE during the 24-week treatment with sovleplenib, only seven (33%) patients had grade 3 events. The most common grade 3 TEAE was anaemia (four [19%] patients), which was not related to treatment. There were no grade 4 or 5 TEAEs. INTERPRETATION:Sovleplenib treatment achieved an encouraging overall haemoglobin response in Chinese patients with warm autoimmune haemolytic anaemia and was well tolerated. The phase 3 part of the study (ESLIM-02) is currently ongoing to further substantiate the efficacy and safety of sovleplenib in this setting. FUNDING:HUTCHMED.

Objective: To investigate the predictive value of soluble FMS-like tyrosine kinase-1 (sFlt-1), coagulation function, and platelet (PLT) parameters for preeclampsia (PE). Methods: A prospective study was conducted on women registered and delivered at Shanghai Fifth People's Hospital from October 2020 to December 2021. All eligible pregnant women were recruited at the time of initial registration in the first trimester. We then obtained serum samples uniformly at 240-280 weeks and stored these samples in a freezer at-80 degrees C and labelled them to create a biobank. Later, when PE was diagnosed, we followed the markers to find their blood samples and complete the tests. Participants were divided into healthy pregnant (HP) and PE groups. Participants were divided into HP and PE groups. Approximately 5 mL of venous blood was collected from each participant at 240-280 weeks gestation. Serum sFlt-1 was measured by enzyme-linked immunosorbent assay. Additionally, D-dimer, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), antithrombin III (ATIII), fibrinogen, PLT, PLT distribution width (PDW), and mean PLT volume (MPV) were recorded. SPSS 27.0 software was used to analyze the correlation of these parameters with PE. Receiver operating characteristic curve analysis determined the optimal cutoff value for each parameter. Results: Serum sFlt-1, APTT, TT, ATIII, PLT, MPV, and PDW levels were significantly different between the PE and HP groups (P < 0.05). Among single-factor indicators for predicting PE, sFlt-1 exhibited the highest value. With an optimal cutoff value of 4.409 ng/mL, sFlt-1 demonstrated a sensitivity and specificity of 85.4% and 87.5%, respectively. The combination of sFlt-1, APTT, TT, PDW, and MPV yielded the highest predictive value, with an area under the receiver operating characteristic curve of 0.946, sensitivity of 86.8%, and specificity of 87.5%. Conclusions: This study demonstrates that a combination of sFlt-1, APTT, TT, PDW, and MPV is a valuable tool for predicting PE.

Abstract Introduction: Primary warm autoimmune hemolytic anemia (pwAIHA) is a rare autoimmune disease leading to accelerated red-cell destruction and anemia. Due to its rarity, the treatment choice is based on personal experience and expert guidelines, and evidence on the treatment response, subsequent relapse and prognosis is not well-understood, especially in China, which are important to inform clinical management of the disease. This study aims to inform evidence-based management for wAIHA by describing the treatment response of adult patients hospitalized with pwAIHA and assessing relapse and survival following treatment. Method: This retrospective observational cohort study utilized electronic medical records (EMR) from three general hospitals in Shanghai including, Huashan Hospital, the Fifth People’s Hospital of Shanghai, and Shanghai General Hospital. Adult patients hospitalized with a confirmed diagnosis of pwAIHA between January 2014 and March 2024 were included. Patients exhibiting evidence of acquired or hereditary causes of hemolytic anemia were excluded from the analysis. Data on demographic, diagnosis history, hemolysis characteristics, treatment and response were collected from the EMR. Relapse, survival and missing information of response were obtained by physicians through post discharge follow-up phone calls until November 15, 2024. A complete response (CR) was defined as normalization of hemoglobin. A partial response (PR) was defined as an increase in hemoglobin > 20 g/L but not normal and absence of transfusion for the previous 7 days. Relapse was defined as a decrease in hemoglobin > 20 g/L after reaching PR, or a reappearance of wAIHA after reaching CR. A logistic regression model was conducted to investigate the factors associated with relapse. The optimal cutoff point for continuous variables were determined and applied in the models. The age-standardized mortality rate was calculated based on the age composition of the 2020 Chinese National Population Census using direct standardization. Results: A total of 104 pwAIHA patients were included, with median age of 63.0 years (IQR 48.8-72.3) at hospitalization and 58.7% comprising female patients. Thirty-nine (37.5%) patients had a previous wAIHA diagnosis and median age was 59.0 years (IQR 44.0-69.3) at the first diagnosis. At study inclusion, a majority of patients (88.3%) had a positive Coombs test. Notably, 84.6% of patients exhibited moderate to severe anemia (<90g/L). Additionally, 85.3%, 91.2% and 84.5% of patients showed elevated reticulocytes (≥ 4%), total bilirubin (≥ 17.1 μmol/L), and lactate dehydrogenase (>250 U/L), respectively, suggesting significant red blood cell destruction. 37.5% of patients had 2 or more comorbidities, with hypertension ranking as the most frequent (18.3%). Almost all patients (94.2%) received corticosteroids as 1L treatment, and only 19 patients advanced to 2L treatment and beyond. Among 93 patients eligible for evaluation, a high response rate (94.6%) is observed with 56 achieved CR and 32 achieved PR, at a median time of 66.0 days (IQR 40.0-145.0) and 14.5 days (IQR 9.0-23.3) after treatment initiation, respectively. Fifty-six patients achieved response had relapse information and about half (53.6%) relapsed, with a median time from response to relapse of 130.0 days (IQR 47.5–480.8). The reticulocyte percentage ≥ 14% at hospitalization (aOR=4.54, p=0.020) was an independent risk indicator for relapse. During follow-up, 33 (31.7%) patients died mostly due to infection (n=21) followed by hemolytic anemia (n=3), cardiovascular disease (n=3) and malignancy (n=3). The age-standardized rate of death was 23.1% (the mortality rate in general population is 0.7%). At 1-, 3- and 5-year post hospitalization, the accumulative death rates were 15.7%, 23.7% and 34.8%, respectively. When considering the whole disease course from 1st diagnosis, the 1-, 3- and 5-year accumulative death rates were 8.9%, 14.3% and 20.3%, respectively. Conclusion: In Chinese patients with pwAIHA, while favorable treatment responses were observed after therapy, there was a notably high rate of relapse accompanied by limited periods of control and poor survival, particularly following hospitalization. This transient relief along with the poor survival outcomes presents a substantial healthcare burden and highlights an urgent need for innovative therapeutic options that can offer more stable and long-term effective treatment for pwAIHA patients in China.

Crovalimab is a novel C5 complement inhibitor that enables rapid and sustained C5 inhibition with subcutaneous, low-volume self-administration every 4 weeks. COMMODORE 2 (NCT04434092) is a global, randomized, open-label, multicenter, phase 3 trial evaluating the non-inferiority of crovalimab versus eculizumab in patients with paroxysmal nocturnal hemoglobinuria not previously treated with C5 inhibition. C5 inhibitor-naive patients with lactate dehydrogenase (LDH) ≥2 × upper limit of normal (ULN) were randomized 2:1 to crovalimab or eculizumab. Co-primary efficacy endpoints were proportion of patients with hemolysis control (centrally assessed LDH ≤1.5 × ULN) and proportion with transfusion avoidance. Secondary efficacy endpoints were proportions of patients with breakthrough hemolysis, stabilized hemoglobin, and change in FACIT-Fatigue score. The primary treatment period was 24 weeks. Two hundred and four patients were randomized (135 crovalimab; 69 eculizumab). Crovalimab was non-inferior to eculizumab in the co-primary endpoints of hemolysis control (79.3% vs. 79.0%; odds ratio, 1.0 [95% CI, 0.6, 1.8]) and transfusion avoidance (65.7% vs. 68.1%; weighted difference, -2.8 [-15.7, 11.1]), and in the secondary efficacy endpoints of breakthrough hemolysis (10.4% vs. 14.5%; weighted difference, -3.9 [-14.8, 5.3]) and hemoglobin stabilization (63.4% vs. 60.9%; weighted difference, 2.2 [-11.4, 16.3]). A clinically meaningful improvement in FACIT-Fatigue score occurred in both arms. Complete terminal complement activity inhibition was generally maintained with crovalimab. The safety profiles of crovalimab and eculizumab were similar with no meningococcal infections. Most patients who switched from eculizumab to crovalimab after the primary treatment period preferred crovalimab. These data demonstrate the positive benefit-risk profile of crovalimab.

Ovarian cancer (OCa) is a common malignancy in women, and the role of cuproptosis and its related genes in OCa is unclear. Using the GSE14407 dataset, we analyzed the expression and correlation of cuproptosis-related genes (CRGs) between tumor and normal groups. From the TCGA-OV dataset, we identified 20 cuproptosis-related long non-coding RNAs (CuLncs) associated with patient survival through univariate Cox analysis. OCa patients were divided into early-stage and late-stage groups to analyze CuLncs expression. Cluster analysis classified patients into two clusters, with Cluster1 having a poorer prognosis. Significant differences in "Lymphatic Invasion" and "Cancer status" were observed between clusters. Seven CRGs showed significant expression differences, validated using the human protein atlas (HPA) databases. Immune analysis revealed a higher ImmuneScore in Cluster1. GSEA identified associated signaling pathways. LASSO regression included 11 CuLncs to construct and validate a survival prediction model, classifying patients into high-risk and low-risk groups. Correlations between riskScore, Cluster phenotype, ImmuneScore, and immune cell infiltration were explored. Cell experiments showed that knocking down AC023644.1 decreases OCa cell viability. In conclusion, we constructed an accurate prognostic model for OCa based on 11 CuLncs, providing a basis for prognosis assessment and potential immunotherapy targets.

Dear Editor, Cytidine analogs,such as decitabine(DAC)and cytarabine(ara-C),have been widely used in the clinical treatment for several can-cer types,including myelodysplastic syndrome and acute mye-loid leukemia(AML;Appelbaum et al.1999;Saba 2007).However,drug resistance causing treatment failure and disease relapse is an unresolved problem to date.

Circular RNAs (circRNAs) have been recently identified as important regulators of various diseases, especially cancer. However, the roles of circRNAs in hematologic malignancies have been rarely reported. This study aimed to identify a specific circRNA expression profile in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and to evaluate the biological roles of circRNA in MDS and AML for understanding their clinical significance. Reverse transcription-quantitative PCR was performed to validate the expression of circZBTB46. Kruskal–Wallis test, Kaplan–Meier curves, and the Cox regression model were employed to analyze the clinical significance of circZBTB46. Two specific shRNAs as well as an expression lentiviral vector of circZBTB46 were constructed to identify the biological function of circZBTB46. The impact of circZBTB46 on leukemia cell proliferation, cell cycle distribution, and apoptosis was confirmed using cell viability assay and flow cytometry analysis. The expression of circZBTB46 gradually increased in patients with higher-risk MDS and AML, as compared to controls. CircZBTB46 expression was significantly correlated with important clinical parameters of MDS, including WHO classification, absolute neutrophil count (ANC), marrow blast, IPSS karyotype, IPSS/IPSS-R risk groups, and AML transformation. CircZBTB46 expression was also associated with ANC, marrow blast, cytogenetic risk groups, FLT3-ITD mutation, and treatment response in AML patients. Furthermore, circZBTB46 overexpression was significantly correlated with shorter overall survival (OS, P = 0.0342, median survival time 18.5 vs. 45.4 months) and leukemia-free survival (LFS, P = 0.0421) in MDS, also with the shorter OS in AML ( P = 0.0293, median survival time 11.6 vs. 16.9 months). Functional studies revealed that silencing circZBTB46 expression significantly inhibited proliferation and induced apoptosis in SKM-1, THP-1, and K562 cell lines, while rescue experiments alleviated the siRNA-mediated growth inhibition and apoptosis in these leukemic cells. The present data suggested the essential oncogenic role of circZBTB46, as a progression and survival indicator in both MDS and AML.

The elderly MDS/AML patients, especially those with TP53 mutations, remain very difficult to treat and resistant to chemotherapy. A total of 105 elderly (age above 60 years) high risk MDS (n = 84, IPSS-R score > 4.5) or AML-MRC (n = 21) patients' records were collected from three medical centers across China and retrospectively analyzed. Decitabine and low dose etoposide was found to be superior to standard decitabine alone in MDS/AML patients with higher ORR (70.0% vs 52.7%) after two cycles of treatment, the longer median durations of ORR (24.0 months vs 12 months) and CR (38.0 months vs 24.0 months), and longer event-free survival (13 months vs 9 months, p = 0.023), without significant improvement in overall survival (20 months vs 15 months, p = 0.059). Other than the TP53 mutation, no other gene mutation (including NRAS, DNMT3A, TET2, EZH2, ASXL1, RUNX1) in the D+E group could reliably predict OS and EFS when compared to wild type phenotype. Patients with TP53 mutations showed higher response rate after two cycles of D+E therapy and had prolonged overall survival (Mutation vs WT: 31 months vs 9 months, p=0.012) and event-free survival (Mutation vs WT: 24 months vs 5 months, p = 0.022) compared with p53 wild type patients in D+E group from multivariate survival analyses. P53 mutated patients receiving D+E treatment also had significantly improved OS (31 months vs 12 months, p = 0.00) and EFS (24 months vs 8 months, p = 0.00) compared to patients receiving decitabine treatment. Moreover, we found decitabine and etoposide greatly reduced TP53 mutant AML tumor burden in the NSG mice xenografts (tumor burden: 92% reduction in p53-null xenograft mice, 65% reduction in p53-wild-type xenograft mice on day 20 after leukemia cells administration, p < 0.0001). It was found that TP53 mutant cells and p53 gene-editing MOLM13 cells showed terminal neutrophil differentiation in the combo of decitabine and etoposide, demonstrating the expected changes in cell-surface CD11b expression and neutrophil functional assays such as phagocytosis. Combo of decitabine and etoposide showed much higher percentage of differentiation marker expression compared to single drug decitabine or etoposide treatment. However, neither the p53 gene-edited THP-1 cells nor the TP53 wild type cell lines displayed markers of terminal differentiation, such as elevated CD11b expression or phagocytosis. Furthermore, the percentage of CD11b-positive cells increased significantly in p53-mutated primary cells while insignificantly in TP53 wild type cells. Decitabine and etoposide induced significant differentiation thus killing p53 mutated myeloid tumor cells and primary patients' cells in vitro. We also uncovered the underlying mechanism that why D+E could induce significant differentiation in TP53 Mutant while not in TP53 wild type myeloid tumors. Notch1 signaling pathway was activated after D+E treatment in TP53 Mutant myeloid tumors, which was consistently correlated with drug-induced myeloid differentiation. Notch1 knockout or Notch inhibitor incubation abrogated drug induced differentiation in TP53 Mutant myeloid tumors. The activation of the Notch1 signaling pathway has been observed to induce disease regression and decrease leukemia stem cells in AML mice as well as significant apoptosis in the AML patient sample. We also found D+E could upregulate transcription factor transcripts such as ETS1, ETS2, which could bind specifically to PSEN1 (PS1) promoter and activate its transcription directly. PS1 represent catalytic subunit of γ-secretase complexes and is responsible for Notch recognition. While in TP53 wild type AML cells, p53 might interact with p300. p300 acts as a coactivator of Ets1 and Ets2, competition for binding to p300 with p53 may confront p300 recruitment and repress ETS transcription activation directly, which might exert its effect on PS1 repression, thus inhibiting γ-secretase complex activity. In short, integrated p53 could repress PSEN1 transcription thus interfering gamma secretase complex activity, Notch recognition and cleavage as well as abrogating drug induced Notch activation and myeloid differentiation, which could be achieved in p53 mutated state. Combination of decitabine and etoposide regimen is also relatively safe and tolerable in elderly patients, which may open up a new path of therapy in the p53 mutated AML/MDS.

Somatic loss-of-function mutations of the dioxygenase Ten-eleven translocation-2 (TET2) occur frequently in individuals with clonal hematopoiesis (CH) and acute myeloid leukemia (AML). These common hematopoietic disorders can be recapitulated in mouse models. However, the underlying mechanisms by which the deficiency in TET2 promotes these disorders remain unclear. Here we show that the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway is activated to mediate the effect of TET2 deficiency in dysregulated hematopoiesis in mouse models. DNA damage arising in Tet2-deficient hematopoietic stem/progenitor cells (HSPCs) leads to activation of the cGAS-STING pathway which in turn promotes the enhanced self-renewal and development of CH. Notably, both pharmacological inhibition and genetic deletion of STING suppresses Tet2 mutation-induced aberrant hematopoiesis. In patient-derived xenograft (PDX) models, STING inhibition specifically attenuates the proliferation of leukemia cells from TET2-mutated individuals. These observations suggest that the development of CH associated with TET2 mutations is powered through chronic inflammation dependent on the activated cGAS-STING pathway and that STING may represent a potential target for intervention of relevant hematopoietic diseases.