A simple protocol to establish a conditionally immortalized mouse podocyte cell line.

Podocytes are specialized terminally differentiated cells in the glomerulus that are the primary target cells in many glomerular diseases. However, the current podocyte cell lines suffer from prolonged in vitro differentiation and limited survival time, which impede research progress. Therefore, it is necessary to establish a cell line that exhibits superior performance and characteristics. We propose a simple protocol to obtain an immortalized mouse podocyte cell (MPC) line from suckling mouse kidneys. Primary podocytes were cultured in vitro and infected with the SV40 tsA58 gene to obtain immortalized MPCs. The podocytes were characterized using Western blotting and quantitative real-time PCR. Podocyte injury was examined using the Cell Counting Kit-8 assay and flow cytometry. First, we successfully isolated an MPC line and identified 39 °C as the optimal differentiation temperature. Compared to undifferentiated MPCs, the expression of WT1 and synaptopodin was upregulated in differentiated MPCs. Second, the MPCs ceased proliferating at a nonpermissive temperature after day 4, and podocyte-specific proteins were expressed normally after at least 15 passages. Finally, podocyte injury models were induced to simulate podocyte injury in vitro. In summary, we provide a simple and popularized protocol to establish a conditionally immortalized MPC, which is a powerful tool for the study of podocytes.

P62 promotes FSH-induced antral follicle formation by directing degradation of ubiquitinated WT1.

In females, the pathophysiological mechanism of poor ovarian response (POR) is not fully understood. Considering the expression level of p62 was significantly reduced in the granulosa cells (GCs) of POR patients, this study focused on identifying the role of the selective autophagy receptor p62 in conducting the effect of follicle-stimulating hormone (FSH) on antral follicles (AFs) formation in female mice. The results showed that p62 in GCs was FSH responsive and that its level increased to a peak and then decreased time-dependently either in ovaries or in GCs after gonadotropin induction in vivo. GC-specific deletion of p62 resulted in subfertility, a significantly reduced number of AFs and irregular estrous cycles, which were same as pathophysiological symptom of POR. By conducting mass spectrum analysis, we found the ubiquitination of proteins was decreased, and autophagic flux was blocked in GCs. Specifically, the level of nonubiquitinated Wilms tumor 1 homolog (WT1), a transcription factor and negative controller of GC differentiation, increased steadily. Co-IP results showed that p62 deletion increased the level of ubiquitin-specific peptidase 5 (USP5), which blocked the ubiquitination of WT1. Furthermore, a joint analysis of RNA-seq and the spatial transcriptome sequencing data showed the expression of steroid metabolic genes and FSH receptors pivotal for GCs differentiation decreased unanimously. Accordingly, the accumulation of WT1 in GCs deficient of p62 decreased steroid hormone levels and reduced FSH responsiveness, while the availability of p62 in GCs simultaneously ensured the degradation of WT1 through the ubiquitin‒proteasome system and autophagolysosomal system. Therefore, p62 in GCs participates in GC differentiation and AF formation in FSH induction by dynamically controlling the degradation of WT1. The findings of the study contributes to further study the pathology of POR.

The single-cell chromatin landscape in gonadal cell lineage specification.

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

Homeobox regulator Wilms Tumour 1 is displaced by androgen receptor at cis-regulatory elements in the endometrium of PCOS patients.

Decidualisation, the process whereby endometrial stromal cells undergo morphological and functional transformation in preparation for trophoblast invasion, is often disrupted in women with polycystic ovary syndrome (PCOS) resulting in complications with pregnancy and/or infertility. The transcription factor Wilms tumour suppressor 1 (WT1) is a key regulator of the decidualization process, which is reduced in patients with PCOS, a complex condition characterized by increased expression of androgen receptor in endometrial cells and high presence of circulating androgens. Using genome-wide chromatin immunoprecipitation approaches on primary human endometrial stromal cells, we identify key genes regulated by WT1 during decidualization, including homeobox transcription factors which are important for regulating cell differentiation. Furthermore, we found that AR in PCOS patients binds to the same DNA regions as WT1 in samples from healthy endometrium, suggesting dysregulation of genes important to decidualisation pathways in PCOS endometrium due to competitive binding between WT1 and AR. Integrating RNA-seq and H3K4me3 and H3K27ac ChIP-seq metadata with our WT1/AR data, we identified a number of key genes involved in immune response and angiogenesis pathways that are dysregulated in PCOS patients. This is likely due to epigenetic alterations at distal enhancer regions allowing AR to recruit cofactors such as MAGEA11, and demonstrates the consequences of AR disruption of WT1 in PCOS endometrium.

Comprehensive Transcriptomic Analysis of EWSR1::WT1 Targets Identifies CDK4/6 Inhibitors as an Effective Therapy for Desmoplastic Small Round Cell Tumors.

Desmoplastic small round cell tumors (DSRCT) are a type of aggressive, pediatric sarcoma characterized by the EWSR1::WT1 fusion oncogene. Targeted therapies for DSRCT have not been developed, and standard multimodal therapy is insufficient, leading to a 5-year survival rate of only 15% to 25%. Here, we depleted EWSR1::WT1 in DSRCT and established its essentiality in vivo. Transcriptomic analysis revealed that EWSR1::WT1 induces unique transcriptional alterations compared with WT1 and other fusion oncoproteins and that EWSR1::WT1 binding directly mediates gene upregulation. The E-KTS isoform of EWSR1::WT1 played a dominant role in transcription, and it bound to the CCND1 promoter and stimulated DSRCT growth through the cyclin D-CDK4/6-RB axis. Treatment with the CDK4/6 inhibitor palbociclib successfully reduced growth in two DSRCT xenograft models. As palbociclib has been approved by the FDA for the treatment of breast cancer, these findings demonstrate the sensitivity of DSRCT to palbociclib and support immediate clinical investigation of palbociclib for treating this aggressive pediatric cancer. SIGNIFICANCE: EWSR1::WT1 is essential for desmoplastic small round cell tumors and upregulates the cyclin D-CDK4/6-RB axis that can be targeted with palbociclib, providing a targeted therapeutic strategy for treating this deadly tumor type.

Overexpression of WT1 in all molecular subtypes of breast cancer and its impact on survival: exploring oncogenic and tumor suppressor roles of distinct WT1 isoforms.

BACKGROUND: Breast cancer is a highly heterogeneous solid tumor, posing challenges in developing targeted therapies effective for all mammary carcinoma subtypes. WT1 emerges as a promising target for breast cancer therapy due to its potential oncogenic role in various cancer types. Previous works have yielded inconsistent results. Therefore, further studies are needed to clarify the behavior of this complex gene in breast cancer. METHODS AND RESULTS: In this study, we examined WT1 expression in both Formalin Fixed Paraffin Embedded breast tumors (n = 41) and healthy adjacent tissues (n = 41) samples from newly diagnosed cases of ductal invasive breast cancer. The fold change in gene expression between the tumor and healthy tissue was determined by calculating 2-∆∆Ct. Disease-free survival analysis was computed using the Kaplan-Meier method. To identify the expression levels of different WT1 isoforms, we explored the ISOexpresso database. Relative quantification of the WT1 gene revealed an overexpression of WT1 in most cases. The percentage of patients surviving free of disease at 8 years of follow-up was lower in the group overexpressing WT1 compared to the group with down-regulated WT1. CONCLUSIONS: Interestingly, this overexpression was observed in all molecular subtypes of invasive breast cancer, underscoring the significance of WT1 as a potential target in all these subtypes. The observed WT1 down-expression in a few cases of invasive breast cancer, associated with better survival outcomes, may correspond to the down-regulation of a particular WT1-KTS (-) isoform: the WT1 A isoform (EX5-/KTS-). The co-expression of this WT1 oncogenic isoform with a regulated WT1- tumor suppressor isoform, such as the major WT1 F isoform (EX5-/KTS +), could also explain such survival outcomes. Due to its capacity to adopt dual roles, it becomes imperative to conduct individual molecular expression profiling of the WT1 gene. Such an approach holds great promise in the development of personalized treatment strategies for breast cancer.

Adjuvant Wilms' tumour 1-specific dendritic cell immunotherapy complementing conventional therapy for paediatric patients with high-grade glioma and diffuse intrinsic pontine glioma: protocol of a monocentric phase I/II clinical trial in Belgium.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) and paediatric high-grade glioma (pHGG) are aggressive glial tumours, for which conventional treatment modalities fall short. Dendritic cell (DC)-based immunotherapy is being investigated as a promising and safe adjuvant therapy. The Wilms' tumour protein (WT1) is a potent target for this type of antigen-specific immunotherapy and is overexpressed in DIPG and pHGG. Based on this, we designed a non-randomised phase I/II trial, assessing the feasibility and safety of WT1 mRNA-loaded DC (WT1/DC) immunotherapy in combination with conventional treatment in pHGG and DIPG. METHODS AND ANALYSIS: 10 paediatric patients with newly diagnosed or pretreated HGG or DIPG were treated according to the trial protocol. The trial protocol consists of leukapheresis of mononuclear cells, the manufacturing of autologous WT1/DC vaccines and the combination of WT1/DC-vaccine immunotherapy with conventional antiglioma treatment. In newly diagnosed patients, this comprises chemoradiation (oral temozolomide 90 mg/m2 daily+radiotherapy 54 Gy in 1.8 Gy fractions) followed by three induction WT1/DC vaccines (8-10×106 cells/vaccine) given on a weekly basis and a chemoimmunotherapy booster phase consisting of six 28-day cycles of oral temozolomide (150-200 mg/m2 on days 1-5) and a WT1/DC vaccine on day 21. In pretreated patients, the induction and booster phase are combined with best possible antiglioma treatment at hand. Primary objectives are to assess the feasibility of the production of mRNA-electroporated WT1/DC vaccines in this patient population and to assess the safety and feasibility of combining conventional antiglioma treatment with the proposed immunotherapy. Secondary objectives are to investigate in vivo immunogenicity of WT1/DC vaccination and to assess disease-specific and general quality of life. ETHICS AND DISSEMINATION: The ethics committee of the Antwerp University Hospital and the University of Antwerp granted ethics approval. Results of the clinical trial will be shared through publication in a peer-reviewed journal and presentations at conferences. TRIAL REGISTRATION NUMBER: NCT04911621.

Wilms' tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP.

In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKÆ´ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment.

Rreb1 is a key transcription factor in Sertoli cell maturation and function and spermatogenesis in mouse.

Spermatogenesis is a developmental process driven by interactions between germ cells and Sertoli cells. This process depends on appropriate gene expression, which might be regulated by transcription factors. This study focused on Rreb1, a zinc finger transcription factor, and explored its function and molecular mechanisms in spermatogenesis in a mouse model. Our results showed that RREB1 was predominantly expressed in the Sertoli cells of the testis. The decreased expression of RREB1 following injection of siRNA caused impaired Sertoli cell development, which was characterized using a defective blood-testis barrier structure and decreased expression of Sertoli cell functional maturity markers; its essential trigger might be SMAD3 destabilization. The decreased expression of RREB1 in mature Sertoli cells influenced the cell structure and function, which resulted in abnormal spermatogenesis, manifested as oligoasthenoteratozoospermia, and we believe RREB1 plays this role by regulating the transcription of Fshr and Wt1. RREB1 has been reported to activate Fshr transcription, and we demonstrated that the knockdown of Rreb1 caused a reduction in follicle-stimulating hormone receptor (FSHR) in the testis, which could be the cause of the increased sperm malformation. Furthermore, we confirmed that RREB1 directly activates Wt1 promoter activity, and RREB1 downregulation induced the decreased expression of Wt1 and its downstream polarity-associated genes Par6b and E-cadherin, which caused increased germ-cell death and reduced sperm number and motility. In conclusion, RREB1 is a key transcription factor essential for Sertoli cell development and function and is required for normal spermatogenesis.

BAP1, Wilms' tumor 1, and calretinin in predicting survival and response to first-line chemotherapy in patients with pleural mesothelioma.

PURPOSE: There are currently no methods to predict response to chemotherapy in pleural mesothelioma (PM). The aim of this study is to investigate the predictive and prognostic role of BAP1, WT1 and calretinin expression and their combinations in pre-treatment tumor samples by immunohistochemical (IHC) staining. METHODS: The study included consecutive PM patients treated with chemotherapy alone at a University hospital between 2009 and 2020. BAP1 analyses were performed on formalin-fixed, paraffin-embedded tumor tissue samples of the patients, while WT1 and calretinin information were obtained from the histopathological diagnosis records. RESULTS: Of the total 107 patients included, 64% had loss of BAP1 expression, whereas 77% had WT1 and 86% had calretinin expression. Patients with the presence of BAP1 expression, one or both of the other two markers, or loss of expression of all three markers (unfavorable status) were more likely to not respond to chemotherapy than those with the presence of all three markers or loss of BAP1 expression and expression of one or two other markers (favorable status) (p = 0.001). Median survival time of patients with favorable and unfavorable status was 15 ± 1.7 and 8.0 ± 2.4 months, respectively (p = 0.027). After adjustment for histopathology and stage, loss of BAP1 (HR = 0.54, 95%CI 0.35-0.83), WT1 (1.75, 1.06-2.90), calretinin (2.09, 1.14-3.84) expression and favourable panel (0.50, 0.27-0.92) was associated with prognosis. CONCLUSIONS: The IHC biomarkers BAP1, WT1, and calretinin, used in the routine diagnosis of PM and their combinations, are the first biomarkers associated with response to chemotherapy and may be a useful tool to select patients for first-line platinum pemetrexed treatment in PM patients. Validation in a large cohort is ongoing.

Wilms' tumor gene 1: lessons from the interface between kidney development and cancer.

In 1990, mutations of the Wilms' tumor-1 gene (WT1), encoding a transcription factor in the embryonic kidney, were found in 10-15% of Wilms' tumors; germline WT1 mutations were associated with hereditary syndromes involving glomerular and reproductive tract dysplasia. For more than three decades, these discoveries prompted investigators to explore the embryonic role of WT1 and the mechanisms by which loss of WT1 leads to malignant transformation. Here, we discuss how alternative splicing of WT1 generates isoforms that act in a context-specific manner to activate or repress target gene transcription. WT1 also regulates posttranscriptional regulation, alters the epigenetic landscape, and activates miRNA expression. WT1 functions at multiple stages of kidney development, including the transition from resting stem cells to committed nephron progenitor, which it primes to respond to WNT9b signals from the ureteric bud. WT1 then drives nephrogenesis by activating WNT4 expression and directing the development of glomerular podocytes. We review the WT1 mutations that account for Denys-Drash syndrome, Frasier syndrome, and WAGR syndrome. Although the WT1 story began with Wilms' tumors, an understanding of the pathways that link aberrant kidney development to malignant transformation still has some important gaps. Loss of WT1 in nephrogenic rests may leave these premalignant clones with inadequate DNA repair enzymes and may disturb the epigenetic landscape. Yet none of these observations provide a complete picture of Wilms' tumor pathogenesis. It appears that the WT1 odyssey is unfinished and still holds a great deal of untilled ground to be explored.

The -KTS splice variant of WT1 is essential for ovarian determination in mice.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

Clinical significance of WT1 in the evaluation of therapeutic effect and prognosis of non-M3 acute myeloid leukemia.

To explore the clinical significance and prognosis of acute myeloid leukemia (AML) patients with WT1 mutations.In total, the clinical data of 269 adult patients with non-M3 AML were considered retrospectively. From these patients, 153 carried WT1 mutation whereas 116 were negative. WT1 mutation positive patients were further divided into WT1 low expression and high expression groups base on the expression level of WT1 by qPCR at diagnosis (cut off: 170500). Survival and therapeutic effect analysis were performed for the above patients with different interfering factors such as co-mutations, the extent of WT1 log reduction and the chemotherapy regimens. Patients with high WT1 expression have higher rate of relapse. We can accurately identify patients with inferior outcomes when we take the following factors into consideration: the WT1 expression level at diagnosis; different prognostic factors including co-mutations (especially NPM1 and FLT3-ITD); the log reduction of WT1 after induction therapy and the risk of stratification. Idarubicin + Cytarabine (IA) regimen could reduce the expression level of WT1 after treatment, and Allo-HSCT played an important role in improving the prognosis of patients with WT1 high expression and patients with WT1 negativity. Among the relapsed patients, there existed a rising trend of WT1-MRD in advance than MFC-MRD and that of patients with continuous complete remission (CR). Different clinical background should be taken into consideration when we judge the prognosis and therapeutic effect of patients with WT1 mutations. In addition, WT1 may be an optional MRD marker, which needs regular monitoring.

Sericin and melatonin mitigate diethylnitrosamine-instigated testicular impairment in mice: Implications of oxidative stress, spermatogenesis, steroidogenesis, and modulation of Nrf2/WT1/SF-1 signaling pathways.

AIMS: This study aimed to investigate the therapeutic influence of combination therapy with sericin and melatonin on attenuating diethylnitrosamine (DEN)-instigated testicular dysfunction in mice and defining the molecular mechanisms involved in orchestrating redox signaling pathways and restoring spermatogenesis and steroidogenesis. MATERIALS AND METHODS: Different groups of male Swiss albino mice were established and injected with respective drugs intraperitoneally. Semen analysis, hormonal assays, and oxidative stress biomarkers were evaluated. Additionally, melatonin and its receptors, WT1, SF-1, vimentin, Nrf2, and ANXA1 expressions were assessed. Histopathological and ultrastructural features of the testes were investigated by semithin, SEM, and TEM analyses. KEY FINDINGS: Exposure to DEN exhibited pathophysiological consequences, including a remarkable increase in lipid peroxidation associated with substantial diminutions in SOD, CAT, GPx, GSH, GSH:GSSG, and GST. Furthermore, it disrupted spermatozoa integrity, testosterone, FSH, LH, melatonin, and its receptors (MT1 and MT2) levels, implying spermatogenesis dysfunction. By contrast, treatment with sericin and melatonin significantly restored these disturbances. Interestingly, the combination therapy of sericin and melatonin noticeably augmented the Nrf2, WT1, and SF-1 expressions compared to DEN-treated mice, deciphering the amelioration perceived in antioxidant defense and spermatogenesis inside cells. Furthermore, immunohistochemical detection of ANXA1 alongside histopathological and ultrastructural analyses revealed evident maintenance of testicular structures without discernible inflammation or anomalies in mice administered with sericin and melatonin compared to the DEN-treated group. SIGNIFICANCE: Our findings highlighted that treatment with sericin and melatonin alleviated the testicular tissues in mice from oxidative stress and dysregulated spermatogenesis and steroidogenesis engendered by DEN.

Biological activities of extracts and compounds from Thai Kae-Lae (Maclura cochinchinensis (Lour.) Corner).

BACKGROUND AND AIMS: The purpose of this study was to investigate the biological properties of Kae-Lae (Maclura cochinchinensis (Lour.) Corner), a traditional medicinal plant used in Ayurvedic recipes in Thailand. To achieve this objective, heartwood samples were collected from 12 sources across Thailand. Fractional extracts (n-hexane, ethyl acetate, and ethanol) and the dominant compounds (morin, resveratrol, and quercetin) were examined for their abilities on cytotoxicity, antioxidant, anti-inflammation, and antileukaemic activity (Wilms' tumour 1 protein was used as a well-known biomarker for leukaemic cell proliferation). METHODS: The study used MTT to assess cytotoxicity in leukaemic cells (K562, EoL-1, and KG-1a). Antioxidant activities were evaluated using ABTS, DPPH, and FRAP assays. The anti-inflammatory activity was investigated by detecting IL-2, TNF-α, and NO using appropriate detection kits. Wilms' tumour 1 protein expression was measured by Western blotting to determine the anti-leukaemic activity. The inhibition of cell migration was also analyzed to confirm anticancer progression. RESULTS: Among the tested extract fraction, ethyl acetate No. 001 displayed strong cytotoxicity specifically in EoL-1 cells, while n-hexane No. 008 demonstrated this effect in three cell lines. Resveratrol, on the other hand, displayed cytotoxicity in all the tested cells. Additionally, the three major compounds, morin, resveratrol, and quercetin, exhibited significant antioxidant and anti-inflammatory properties. In particular, resveratrol demonstrated a noteworthy decreased Wilms' tumour 1 protein expression and a reduction in cell proliferation across all cells. Moreover, ethyl acetate No. 001, morin, and resveratrol effectively inhibited MCF-7 cell migration. None of these compounds showed any impact on red blood cell haemolysis. CONCLUSION: Based on these findings, it can be concluded that Kae-Lae has promising chemotherapeutic potential against leukaemic cells, with fractional extracts (ethyl acetate and n-hexane) and resveratrol exhibiting the most potent cytotoxic, antioxidant, anti-inflammatory, and anti-cell migration activities.

Prognostic value of combined WT1 and multiparameter flow cytometry assessment for measurable residual disease after induction in non-APL acute myeloid leukemia.

The objective of this study was to investigate the expression pattern of Wilms tumor 1 (WT1) gene at diagnosis, complete remission (CR) and relapse status in non-acute promyelocytic leukemia (non-APL) acute myeloid leukemia (AML) patients, and further explore the prognostic value of measurable residual disease (MRD) assessment by WT1 gene and multiparameter flow cytometry (MFC). Our results showed that the average expression level of WT1 was 4026 ± 616.1 copies/104 ABL at diagnosis, 155.3 ± 36.03 copies/104 ABL at CR, and 1766 ± 238.8 copies/104 ABL at relapse, with statistically significant differences (p = .000). ROC analysis showed that WT1 expression levels were 118.1 copies/104 ABL and MFC-MRD was 0.155%, which had good predictive efficacy for relapse of patients during consolidation therapy. Both WT1-MRD and MFC-MRD had a significant impact on relapse-free survival (RFS) and overall survival (OS). Patients with WT1-MRD positive or MFC-MRD positive were associated with worse RFS (HR 3.840, 95% CI 1.582-9.320, p = .003), (HR 4.464, 95% CI 1.841-10.984, p = .001) and worse OS (HR 2.963, 95% CI 1.058-8.295, p = .039), (HR 3.590, 95% CI 1.254-10.280, p = .017). Besides, compared with patients who were negative for both WT1-MRD and MFC-MRD, patients who were positive both WT1-MRD and MFC-MRD were associated with worse RFS (HR 6.200, 95% CI 2.206-17.430, p = .001) and worse OS (HR 4.886, 95% CI 1.388-17.197, p = .013). This study demonstrates that combined assessment of MRD by WT1 and MFC improves relapse and prognosis prediction in non-APL AML patients, and may help guide interventions for disease relapse.

The miR-143/145 cluster induced by TGF-ß1 suppresses Wilms' tumor 1 expression in cultured human podocytes.

Transforming growth factor (TGF)-ß1 contributes to podocyte injury in various glomerular diseases, including diabetic kidney disease, probably at least in part by attenuating the expression of Wilms' tumor 1 (WT1). However, the precise mechanisms remain to be defined. We performed miRNA microarray analysis in a human podocyte cell line cultured with TGF-ß1 to examine the roles of miRNAs in podocyte damage. The microarray analysis identified miR-143-3p as the miRNA with the greatest increase following exposure to TGF-ß1. Quantitative RT-PCR confirmed a significant increase in the miR-143-3p/145-5p cluster in TGF-ß1-supplemented cultured podocytes and demonstrated upregulation of miR-143-3p in the glomeruli of mice with type 2 diabetes. Ectopic expression of miR-143-3p and miR-145-5p suppressed WT1 expression in cultured podocytes. Furthermore, inhibition of Smad or mammalian target of rapamycin signaling each partially reversed the TGF-ß1-induced increase in miR-143-3p/145-5p and decrease in WT1. In conclusion, TGF-ß1 induces expression of miR-143-3p/145-5p in part through Smad and mammalian target of rapamycin pathways, and miR-143-3p/145-5p reduces expression of WT1 in cultured human podocytes. miR-143-3p/145-5p may contribute to TGF-ß1-induced podocyte injury.NEW & NOTEWORTHY This study by miRNA microarray analysis demonstrated that miR-143-3p expression was upregulated in cultured human podocytes following exposure to transforming growth factor (TGF)-ß1. Furthermore, we report that the miR-143/145 cluster contributes to decreased expression of Wilms' tumor 1, which represents a possible mechanism for podocyte injury induced by TGF-ß1. This study is important because it presents a novel mechanism for TGF-ß-associated glomerular diseases, including diabetic kidney disease (DKD), and suggests potential therapeutic strategies targeting miR-143-3p/145-5p.

WT1+ glomerular parietal epithelial progenitors promote renal proximal tubule regeneration after severe acute kidney injury.

Rationale: Mammalian renal proximal tubules can partially regenerate after acute kidney injury (AKI). However, cells participating in the renal proximal tubule regeneration remain to be elucidated. Wilms' tumor 1 (WT1) expresses in a subtype of glomeruli parietal epithelial cells (PECs) in adult kidneys, it remains unclear whether these WT1+ PECs play a role in renal regeneration/repair after AKI. Methods: Ischemia-reperfusion injury (IRI) mouse model was used to investigate the expression pattern of WT1 in the kidney after severe AKI. Conditional deletion of WT1 gene mice were generated using Pax8CreERT2 and WT1fl/fl mice to examine the function of WT1. Then, genetic cell lineage tracing and single-cell RNA sequencing were performed to illustrate that WT1+ PECs develop into WT1+ proximal tubular epithelial cells (PTECs). Furthermore, in vitro clonogenicity, direct differentiation analysis and in vivo transplantation were used to reveal the stem cell-like properties of these WT1+ PECs. Results: The expression of WT1 protein in PECs and PTECs was increased after severe AKI. Conditional deletion of WT1 gene in PTECs and PECs aggravated renal tubular injury after severe AKI. WT1+ PECs develop into WT1+ PTECs via the transient scattered tubular cell stage, and these WT1+ PECs possess specific stem cell-like properties. Conclusions: We discovered a group of WT1+ PECs that promote renal proximal tubule regeneration/repair after severe AKI, and the expression of WT1 in PECs and PTECs is essential for renal proximal tubule regeneration after severe kidney injury.

The people behind the papers - Marina Ramiro-Pareta and Ofelia Martinez-Estrada.

Wilms tumor 1 (WT1) is a transcription factor known to be expressed in the epicardium and required for heart development, but the role of WT1 outside of the epicardium is less clear. In a new paper in Development, Marina Ramiro-Pareta and colleagues generate an inducible, tissue-specific loss-of-function mouse model to investigate the role of WT1 in coronary endothelial cells (ECs). We caught up with first author Marina Ramiro-Pareta and corresponding author Ofelia Martinez-Estrada (Principal Investigator at the Institute of Biomedicine in Barcelona, Spain) to learn more about their research.

Wilms' tumor 1 expression combined with genetic mutations for prognostic assessment in MDS.

Overexpression of Wilms' tumor (WT1) is frequently observed in myelodysplastic syndrome (MDS), which has been proposed as a prognostic marker. However, the prognostic role of WT1 expression in different contexts remains to be fully elucidated. We retrospectively assessed the relationships between WT1 levels and preexisting prognostic factors to further investigate its prognostic role under different contexts. In our study, WT1 expression was positively correlated with WHO 2016 classification and IPSS-R stratification. Lower WT1 expression was found in relation to TET2, TP53, CD101, or SRSF2 mutations, while mutant NPM1 patients possessed higher level. Notably, WT1 overexpression maintained its inferior prognostic effect on overall survival (OS) in TP53-wild patients but not in TP53-mutated group. In multivariate analysis, higher WT1 expression was a risk factors for OS in EB patients without TP53 mutations. Overall, WT1 expression was useful to predict prognosis for MDS and its prognostic role was impacted by some gene mutations.