Wilms Tumor 1 Research Articles

Kidney Organoid Modeling of WT1 Mutations Reveals Key Regulatory Paths Underlying Podocyte Development.

Wilms tumor-1(WT1) is a crucial transcription factor that regulates podocyte development. However, the epigenomic mechanism underlying the function of WT1 during podocyte development has yet to be fully elucidated. Here, single-cell chromatin accessibilityand gene expressionmaps of foetal kidneys and kidney organoids are generated. Functional implications of WT1-targeted genes, which are crucial for the development of podocytes and the maintenance of their structure, including BMPER/PAX2/MAGI2 that regulates WNT signaling pathway, MYH9 that maintains actin filament organization and NPHS1 that modulates cell junction assembly are identified. To further illustrate the functional importance of WT1-mediated transcriptional regulation during podocyte development, cultured and implanted patient-derived kidney organoids derived from the Induced Pluripotent Stem Cell (iPSCs) of a patient with a heterozygous missense mutation in WT1 are generated. Results from single-cell RNA sequencing (scRNA-seq)and functional assays confirm that the WT1 mutation leads to delays in podocyte development and causes damage to cell structures, due to its failure to activate the targeting genes MAGI2, MYH9, and NPHS1. Notably, correcting the mutation in the patient iPSCs using CRISPR-Cas9 gene editing rescues the podocyte phenotype. Collectively, this work elucidates the WT1-related epigenomic landscape with respect to human podocyte development and identifies the disease-causing role of a WT1 mutation.

Photosynthetic Bacteria-Hitchhiking 2D iMXene-mRNA Vaccine to Enable Photo-Immunogene Cancer Therapy.

Therapeutic mRNA vaccines have become powerful therapeutic tools for severe diseases, including infectious diseases and malignant neoplasms. mRNA vaccines encoding tumor-associated antigens provide unprecedented hope for many immunotherapies that have hit the bottleneck. However, the application of mRNA vaccines is limited because of biological instability, innate immunogenicity, and ineffective delivery in vivo. This study aims to construct a novel mRNA vaccine delivery nanosystem to successfully co-deliver a tumor-associated antigen (TAA) encoded by the Wilms' tumor 1 (WT1) mRNA. In this system, named PSB@Nb1.33C/mRNA, photosynthetic bacteria (PSB) efficiently delivers the iMXene-WT1 mRNA to the core tumor region using photo-driven and hypoxia-driven properties. The excellent photothermal therapeutic (PTT) properties of PSB and 2D iMxene (Nb1.33C) trigger tumor immunogenic cell death, which boosts the release of the WT1 mRNA. The released WT1 mRNA is translated, presenting the TAA and amplifying immune effect in vivo. The designed therapeutic strategy demonstrates an excellent ability to inhibit distant tumors and counteract postsurgical lung metastasis. Thus, this study provides an innovative and effective paradigm for tumor immunotherapy, i.e., photo-immunogene cancer therapy, and establishes an efficient delivery platform for mRNA vaccines, thereby opening a new path for the wide application of mRNA vaccines.

CD8+ T cell targeting of tumor antigens presented by HLA-E.

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E-restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E-restricted CD8+ T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E-restricted CD8+ T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E+ prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8+ T cell-based immunotherapies.

Palisaded Neutrophilic and Granulomatous Dermatitis-Like Reaction Associated with Wilms Tumor 1 Protein-Derived Peptide Vaccine: A Case and Review of the Literature

Palisaded neutrophilic and granulomatous dermatitis (PNGD) is an inflammatory cutaneous reaction that is associated with underlying autoimmune diseases. In rare cases, PNGD has been attributed to systemic medications. Here we report one case of a cutaneous injection-site reaction that occurred after intradermal administration of an experimental Wilms Tumor 1 (WT1) protein-derived peptide vaccine in a woman being treated for recurrent ovarian cancer. This is the first reported case of a PNGD-like reaction associated with immunotherapy injections. In this article, we discuss this case and summarize the literature related to the diagnosis and management of PNGD, as well as other injection site reactions related to immunotherapies and experimental vaccines.

Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML

BackgroundHematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due...

Decade-long WT1-specific CTLs induced by WT1 peptide vaccination.

The peptide-based cancer vaccine targeting Wilms' tumor 1 (WT1) is a promising immunotherapeutic strategy for hematological malignancies. It remains unclear how long and to what extent the WT1-specific CD8 + cytotoxic T cell (CTL) persist after WT1 peptide vaccination. The WT1 peptide vaccine was administered with written consent to a patient with CML in the chronic phase who did not respond well to imatinib, and the patient was followed for 12 years after vaccination. Immune monitoring was performed by specific amplification of WT1-specific CTLs using a mixed lymphocyte peptide culture. T-cell receptors (TCRs) of amplified WT1-specific CTLs were analyzed using next-generation sequencing. This study was approved by the Institutional Review Board of our institution. WT1-specific CTLs, which were initially detected during WT1 peptide vaccination, persisted at a frequency of less than 5 cells per 1,000,000 CD8 + T cells for more than 10 years. TCR repertoire analysis confirmed the diversity of WT1-specific CTLs 11 years after vaccination. CTLs exhibited WT1 peptide-specific cytotoxicity in vitro. The WT1 peptide vaccine induced an immune response that persists for more than 10 years, even after cessation of vaccination in the CML patient.

New-onset diabetes after kidney transplantation: Assessing urinary Wilm's tumor-1 protein to predict renal allograft dysfunction

PurposeNew-onset diabetes after transplantation (NODAT) is a frequent metabolic complication associated with podocyte damage and renal allograft dysfunction. Thus, Wilm's tumor-1 (WT-1) protein, as a podocyte marker, holds promise as an option to evaluate renal allograft dysfunction in NODAT. Therefore, the study aimed to investigate urinary WT-1 levels in NODAT patients during the first year after kidney transplantation (KTx). Materials and methodsKTx patients were categorized into non-NODAT and NODAT groups. Fasting blood glucose, glycated hemoglobin (HbA1c), urinary albumin/creatinine ratio (ACR), serum creatinine, estimated glomerular filtration rate (eGFR), and urinary WT-1 were measured at 3, 6, 9, and 12-months post-KTx. ResultsThe NODAT group manifested elevated levels of blood glucose and HbA1c during the first year post-KTx. Also, exhibited elevations in ACR and serum creatinine levels at 6, 9, and 12-months post-KTx when compared to non-NODAT group. Conversely, eGFR values in the NODAT group demonstrated significant declines at 3, 6, and 9-months post-KTx relative to non-NODAT. Furthermore, NODAT group exhibited a median annual eGFR of 47 ​mL/min/1.73 ​m2. Urinary WT-1 levels at 3, 6, 9, and 12-months post-KTx were significantly higher in the NODAT group compared to non-NODAT. Additionally, noteworthy positive correlations were identified between urinary WT-1 and HbA1c levels, along with significant negative correlations between urinary WT-1 and eGFR at the 3, 6, 9, and 12-months post-KTx. ConclusionThe increased urinary WT-1 levels from 3-months post-KTx in NODAT patients may indicate the first sign of podocyte injury, predicting a renal allograft dysfunction in these patients.

GATA2 mutant variant allele frequency may reflect prognosis in Chinese adult patients with de novo cytogenetically normal acute myeloid leukemia.

Exploration of variant allele frequency (VAF) of GATA2 mutations (GATA2mut) provides insights into acute myeloid leukemia (AML) prognosis. In this study, we analyzed GATA2mut and co-mutations in 166 Chinese patients with cytogenetically normal AML. This was done through targeted next-generation sequencing of 34 genes associated with myeloid leukemia. GATA2mut was identified in 17 (10%) patients being significantly correlated with co-mutations in CCAAT/enhancer-binding protein alpha (CEBPA) double mutation (P = 0.001). We observed that the N-terminal zinc finger domain (ZF1) was linked to CEBPA mutations, while the C-terminal zinc finger domain (ZF2) was associated with Wilms' tumor 1 (WT1) mutations. It was also noted that patients with GATA2mut had lower platelet counts at diagnosis (P = 0.032). In the entire cohort, GATA2mut had no significant prognostic impact on overall survival (OS) (P = 0.762) and relapse-free survival (RFS) (P = 0.369) compared to patients with GATA2wt. The OS (P = 0.737) and RFS (P = 0.894) of the ZF1 mutation were similar to those of the ZF2 mutation. Most patients with GATA2 mutations were classified in the ELN2022 favorable- and intermediate-risk groups. GATA2mut patients in the favorable-risk group were divided into GATA2High and GATA2Low groups using a median cutoff variant allele frequency (VAF) of 40.13%. GATA2High patients were associated with worse OS (P = 0.031) and RFS (P = 0.021) than GATA2Low patients. In the intermediate-riskgroup, the high median VAF ofGATA2 (≥38.51%) had no significant effect in OS and RFS compared with the low median VAF (<38.51%). This study offers new insights on the prognosis of GATA2mut in the favorable-risk group, where VAF can be used as a guide.

WT1 and TP53 as valuable diagnostic biomarkers for relapse after hematopoietic stem cell transplantation in acute myeloid leukemia.

Relapse following hematopoietic stem cell transplantation (HSCT) occurs relatively frequently and is a significant risk factor for mortality in patients with acute myeloid leukemia (AML). Early diagnosis is, therefore, of utmost importance and can provide valuable guidance for appropriate and timely intervention. Here, the diagnostic value of two molecular markers, Wilms tumor 1 (WT1) and tumor suppressor protein p53 (TP53), were studied. Twenty AML patients undergoing HSCT participated in this investigation. Some had relapsed following HSCT, while others were in remission. Peripheral blood (PB) and bone marrow (BM) samples were collected following relapse and remission. WT1 and TP53 messenger RNA (mRNA) expression was evaluated using reverse transcription-quantitative polymerase chain reaction (RT‒qPCR). The diagnostic value of genes was evaluated by utilizing receiver-operating characteristic (ROC) curve analysis. ROC analysis showed WT1 and TP53 as diagnostic markers for relapse after HSCT in AML patients. The mRNA expression level of WT1 was elevated in individuals who experienced relapse compared to those in a state of remission (p value < 0.01). Conversely, the expression level of TP53 mRNA was lower in individuals who had relapsed compared to those in remission (p value < 0.01). WT1 and TP53 possess the potential to serve as invaluable biomarkers in the identification of molecular relapse after HSCT in patients with AML. Further studies for a definitive conclusion are recommended.

Prognostic significance of Wilms' tumor gene 1 expression in children with B-cell precursor acute lymphoblastic leukemia.

The prognostic role of Wilms' tumor 1 (WT1) gene expression at diagnosis in children with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is still controversial. We detected the WT1 transcript levels of 533 de novo pediatric BCP-ALL patients using TaqMan-based real-time quantitative PCR and analyzed their clinical features. The WT1 transcript levels differed among the distinct molecularly defined groups, with the highest levels in the KMT2A rearrangements (KMT2A-r) group. According to the results of the X-tile software, all patients were divided into two groups: WT1/ABL ≥ 0.24% (group A) and <0.24% (group B). The proportions of patients whose age was ≥10 years old, with immunophenotype of Pro-B, belonging in high-risk group, or with minimal residual disease (MRD) ≥ 0.01% at week 12 were significantly higher in group A than in group B. In the B-other group, WT1 overexpression was an independent risk factor of overall survival (OS) rate (P = 0.042), and higher MRD ≥ 0.01% at week 12 was associated with lower OS rate (P<0.001) and event-free survival rate (P<0.001). Moreover, the subgroup analysis revealed that, in patients with initial WBC<50 × 109/L or MRD<0.1% at day 33 or MRD<0.01% at week 12 or in the standard-risk group, WT1 overexpression led to a poorer outcome in comparison with those with WT1 downexpression (P<0.05). Therefore, pediatric BCP-ALL with WT1 overexpression had unique clinico-pathological characteristics and poor treatment response. In B-other patients, WT1 overexpression at diagnosis predicted an inferior prognosis. The WT1 gene may serve as a biomarker for monitoring residual disease in the B-other population, especially in children in the standard-risk group.

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 IKKƴ 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.

Enhancing the immunogenicity of Wilms tumor 1 epitope in mesothelioma cells with immunoproteasome inhibitors.

The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit β5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes.

Network pharmacology and molecular docking of endogenous active metabolites in diabetic kidney disease

Objectives Network pharmacology and molecular docking were used to predict endogenous active metabolites with protective effects in diabetic kidney disease (DKD). Methods We utilized metabolomics to screen differentially expressed metabolites in kidney tissues of mice with type 2 DKD and predicted potential targets using relevant databases. The interaction network between endogenous active metabolites and target proteins was established by integrating differentially expressed metabolites and proteins associated with DKD identified through proteomics. Gene ontology (GO) and signaling pathway enrichment analysis were performed. The biological functions of the active candidate metabolites and their effects on downstream pathways were also verified. Results Metabolomics revealed 130 differentially expressed metabolites. Through co-expression network analysis coupled with the investigation of differentially expressed proteins in proteomics, 2-hydroxyphenylpropionylglycine (2-HPG) emerged as a key regulator of DKD. 2-HPG was found to modulate the progression of DKD by regulating the conformation and activity of synaptophysin 1 (SYNJ1), with a correlation coefficient of 0.974. In vivo experiments revealed that SYNJ1 expression was significantly downregulated in the Macroalbuminuria Group compared to the Control Group and negatively correlated with proteinuria (r = −0.7137), indicating its important role in DKD progression. Immunofluorescence demonstrated that treatment with 2-HPG restores the expression of the foot process marker protein Wilms tumor-1 (WT-1) in podocytes injured by high glucose levels. Western blot and polymerase chain reaction support the involvement of SYNJ1 in this process. Conclusions This study demonstrated the significance of the 2-HPG/SYNJ1 signaling axis in safeguarding the foot process of podocytes in DKD.

Evaluation of glomerular sirtuin-1 and claudin-1 in the pathophysiology of nondiabetic focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is the leading cause of nephrotic syndrome, which is characterized by podocyte injury. Given that the pathophysiology of nondiabetic glomerulosclerosis is poorly understood and targeted therapies to prevent glomerular disease are lacking, we decided to investigate the tight junction protein claudin-1 and the histone deacetylase sirtuin-1 (SIRT1), which are known to be involved in podocyte injury. For this purpose, we first examined SIRT1, claudin-1 and podocin expression in kidney biopsies from patients diagnosed with nondiabetic FSGS and found that upregulation of glomerular claudin-1 accompanies a significant reduction in glomerular SIRT1 and podocin levels. From this, we investigated whether a small molecule activator of SIRT1, SRT1720, could delay the onset of FSGS in an animal model of adriamycin (ADR)-induced nephropathy; 14 days of treatment with SRT1720 attenuated glomerulosclerosis progression and albuminuria, prevented transcription factor Wilms tumor 1 (WT1) downregulation and increased glomerular claudin-1 in the ADR + SRT1720 group. Thus, we evaluated the effect of ADR and/or SRT1720 in cultured mouse podocytes. The results showed that ADR [1 µM] triggered an increase in claudin-1 expression after 30 min, and this effect was attenuated by pretreatment of podocytes with SRT1720 [5 µM]. ADR [1 µM] also led to changes in the localization of SIRT1 and claudin-1 in these cells, which could be associated with podocyte injury. Although the use of specific agonists such as SRT1720 presents some benefits in glomerular function, their underlying mechanisms still need to be further explored for therapeutic use. Taken together, our data indicate that SIRT1 and claudin-1 are relevant for the pathophysiology of nondiabetic FSGS.

Blockade of Notch1 Signaling Alleviated Podocyte Injury in Lupus Nephritis Via Inhibition of NLRP3 Inflammasome Activation.

To explore the role of Notch1 pathway in the pathogenesis of podocyte injury, and to provide novel strategy for podocyte repair in lupus nephritis (LN). Bioinformatics analysis and immunofluorescence assay were applied to determine the expression and localization of Notch1 intracellular domain1 (NICD1) in kidneys of LN patients and MRL/lpr mice. The stable podocyte injury model in vitro was established by puromycin aminonucleoside (PAN) treatment. Expression of inflammasome activation related gene was detected by qPCR. The podocytes with PAN treatment were cultured with or without N-S-phenyl-glycine-t-butylester (DAPT), an inhibitor of Notch1 pathway. NICD1, Wilm'stumor1 (WT1), nucleotide-binding oligomerization domain-like receptors 3 (NLRP3), and absent in melanoma-like receptors 2 (AIM2) were detected by western blot. In vivo, MRL/lpr mice were administrated with DAPT or vehicle. The LN symptoms were assessed. The podocyte injury was evaluated, and the NLRP3 in podocytes of mice was detected. Notch1 pathway was overactivated in glomeruli of LN patients. NICD1 was colocalized with podocytes of LN patients and MRL/lpr mice. The inflammasome-related genes were significantly increased in podocytes with PAN treatment. NICD1 and NLRP3 were significantly decreased, while WT1 was significantly increased in injured podocytes treated with DAPT in vitro. In vivo, lupus-like symptoms were alleviated in DAPT treatment group. Notch1 pathway was inhibited in kidneys of mice treated with DAPT. The renal inflammation was reduced and the podocyte injury was mitigated in DAPT treatment group. The NLRP3 was decreased in podocytes of mice treated with DAPT. Notch1 pathway was overactivated in podocytes of LN patients and MRL/lpr mice. Blockade of Notch1 pathway reduced renal inflammation and alleviated podocyte injury via inhibition of NLRP3 inflammasome activation in LN.

10243-IMT-3 LONG-TERM OUTCOME OF A COCKTAIL WILMS' TUMOR 1 (WT1) VACCINATION USING TWO HLA CLASS I PEPTIDES AND ONE CLASS II PEPTIDE AGAINST RECURRENT MALIGNANT GLIOMAS

Abstract PURPOSE Our clinical trials show the safety and clinical efficacy of Wilms' tumor 1 (WT1) human leukocyte antigen (HLA) class I (Izumoto S et al. J Neurosurg. 2008) and class II (Tsuboi A et al. Cancer Immunol Immunother. 2019) peptide vaccination for recurrent malignant gliomas have been established. We have developed a cocktail vaccine (WT1 trio) containing two class I peptides (HLA-A*24:02 and HLA-A*02:01) and one II class peptide to improve more effective immunological response and improve patient's prognosis. Clinical trial of a cocktail vaccination using WT1 HLA class I and II peptides for recurrent malignant gliomas is planned to verify its safety, clinical efficacy and usefulness of surrogate markers. Patients and METHODS Twenty-three patients with recurrent malignant gliomas, which showed WT1-positive in tumor samples and HLA-A*24:02 or HLA-A*02:01-positive in blood sample, were enrolled. These patients (age: 26-72 years old, average: 49.4) included 15 cases of glioblastomas and 8 of anaplastic astrocytomas. Patients received a WT1 trio vaccine intradermally, 7 times at 2-week intervals during 3 months.WT1-DTH and WT1-IgG antibody were regularly measured. Vaccine-related adverse events, best clinical response and the transfer rate of long-term administration of WT1 trio vaccination were estimated. RESULTS WT1-DTH positive cases were 12, WT1-IgG antibody positive were in 11. In most patients, WT1 -DTH positiveness coincided with that of WT1-IgG antibody. 9 of 11 cases showed stable disease at 3 months and transferred long-term administration of WT1 trio vaccination. Transfer rate in GBM and AA of long-term administration was 33% and 25%, respectively. Grade1 skin eruption was observed at the injection sites in 15 cases, but no significant adverse events related with vaccination were shown. Conclusion: WT1-DTH and WT1-IgG antibody may be useful surrogate markers. Long-term outcome of WT1 trio vaccination was verified for recurrent malignant gliomas.

MicroRNA193a: An Emerging Mediator of Glomerular Diseases.

MicroRNAs (miRNAs) are noncoding small RNAs that regulate the protein expression of coding messenger RNAs. They are used as biomarkers to aid in diagnosing, prognosticating, and surveillance of diseases, especially solid cancers. MiR-193a was shown to be directly pathogenic in an experimental mouse model of focal segmental glomerulosclerosis (FSGS) during the last decade. Its specific binding and downregulation of Wilm's tumor-1 (WT-1), a transcription factor regulating podocyte phenotype, is documented. Also, miR-193a is a regulator switch causing the transdifferentiation of glomerular parietal epithelial cells to a podocyte phenotype in in vitro study. Interaction between miR-193a and apolipoprotein 1 (APOL1) mRNA in glomeruli (filtration units of kidneys) is potentially involved in the pathogenesis of common glomerular diseases. Since the last decade, there has been an increasing interest in the role of miR-193a in glomerular diseases, including diabetic nephropathy and membranous nephropathy, besides FSGS. Considering the lack of biomarkers to manage FSGS and diabetic nephropathy clinically, it is worthwhile to invest in evaluating miR-193a in the pathogenesis of these diseases. What causes the upregulation of miR-193a in FSGS and how the mechanism is different in different glomerular disorders still need to be elucidated. This narrative review highlights the pathogenic mechanisms of miR-193a elevation in various glomerular diseases and its potential use in clinical management.

Wilms' Tumor 1 Functions As a Tumor Suppressor to Suppress FLT3-STAT Signaling and Epigenetic Remodeling in Acute Myeloid Leukemia (CALGB 8461, 9665 and 20202; Alliance)

The Wilms' Tumor 1 ( WT1) gene is a transcription factor that is recurrently mutated or commonly overexpressed in several cancer types. In acute myeloid leukemia (AML), frequent overexpression of WT1 and poor patient outcomes associated with WT1 mutations highlight its importance in the disease; however, there are no tailored treatments for these patients. Furthermore, WT1's fundamental role as either an oncogene or tumor-suppressor remains unresolved. Here we examine the roles of both wild-type and mutant WT1 in AML through epigenetic and mechanistic studies. Using our findings we propose a personalized treatment strategy. WT1 mutations are enriched in the NPM1 mut subset of AML; thus we first focused our study on a cohort of 581 patients with de novo AML and NPM1 mutations enrolled on Alliance for Clinical Trials in Oncology studies. Transcriptomic analyses revealed that WT1 mut patientsphenocopied a distinct, aberrant gene expression signature associated with FLT3 internal tandem duplications (ITD), a mutation known to activate STAT signaling and associated with poor outcome in AML. We observed that WT1 expression levels were remarkably elevated in FLT3-ITD patients and were driven by STAT5A binding to the WT1 promoter. STAT5A binding and subsequent WT1 upregulation were blocked by small molecule FLT3 inhibitors. These findings linking FLT3 activity with WT1 expression raise two possibilities: either WT1 is an oncogene cooperating with the FLT3-STAT pathway, or WT1 naturally functions to suppress FLT3 signaling in a negative feedback loop subverted by WT1 mutations. As WT1 interacts with TET2 to facilitate epigenetic remodeling and DNA binding, we performed genome-wide DNA methylation analysis on WT1 mut AML patients and observed selective hypermethylation of WT1 binding motifs consistent with loss of function. We confirmed that WT1 mutations in AML commonly cause truncation of the C-terminal DNA binding domain in our patient cohort. Using co-immunoprecipitation, ChIP-sequencing and luciferase reporter assays, we found that mutant WT1 functions as a dominant-negative, inhibiting WT1 derived from the wild-type allele. Investigation of WT1 target genes by intersecting transcriptomic and DNA methylation profiles identified hypermethylation and downregulation of miR-193a, leading to a significant downregulation of miR-193a-3p in WT1 mut AML patients. Overexpression of wild-type, but not mutated, WT1 rescued miR-193a expression in AML cell lines. Enforced expression of miR-193a significantly delayed AML onset in vivo. In addition, overexpression of miR-193a in multiple human AML cell lines and primary patient samples impaired AML cell growth and colony-forming capacity while promoting monocytic differentiation, underscoring its role as potent tumor suppressor. Therapeutic modulation of miRNA levels in cancer patients has been limited by inefficient delivery and tissue enrichment. To overcome this, we tested a novel lipid-nanoparticle (LNP) formulation of miR-193a-3p (INT-1B3), currently being investigated in a phase I clinical trial (NCT04675996). Biweekly i.v. treatments of INT-1B3 in the immunocompetent Hoxa9/Meis1 (H9M)-transduced model system prevented AML formation, highlighting the potent anti-leukemic activity of this miRNA based therapeutic. Overexpression of miR-193a-loaded LNPs downregulated FLT3 expression and suppressed STAT signaling in primary AML samples. Finally, treatment of primary AML cells with FLT3 inhibitors revealed enhanced sensitivity of WT1 mut cells in the absence of FLT3-ITD, highlighting the role of wild-type FLT3 in WT1 mut cells. In summary, we uncovered a critical negative-feedback loop maintained by WT1 to suppress FLT3 activity. Loss-of-function WT1 mutations subvert the tumor suppressor function of WT1 via failure to maintain miR-193a expression, leading to increased FLT3 expression and STAT5 signaling, subsequently impairing differentiation, increasing proliferation and disease aggressiveness in AML (Figure 1). Our findings advocate for use of FLT3 inhibition and miR-193a supplementation for treatment of WT1 mut patients, a subgroup with poor outcomes and no targeted treatment options.

NGS Profile and the Mathematical Prediction Model for Venetoclax Combination Therapy in HM-Screen-Japan 02 Study

[Introduction] Azacitidine and venetoclax combination therapy (Aza/Ven) is a novel strategy for acute myeloid leukemia (AML). Although this regimen is widely used as a first line treatment not only for unfit/elderly patients but also for fit/young patients in the real-world situation, appropriate patient selection is an important issue. HM-SCREEN-Japan 02 (UMIN-CTR UMIN000046371) is a Japanese multicenter prospective observational study to evaluate the clinical utility of targeted sequencing. In this study, we performed the sub-analysis of newly diagnosed AML patients who received Aza/Ven as a 1 st line treatment to elucidate the genetic background of AML. Furthermore, we proposed a novel mathematical model to predict the response of Aza/Ven. [Methods] AmoyDx® Myeloid Blood Cancer Panel for acute myeloid leukemia was used for the targeted sequencing. We performed linear discriminant analysis to predict complete response patients by Aza/Ven based on clinical information before treatment. We utilized three clinical quantities for the analysis: Wilms Tumor-1 (WT1) mRNA expression level, the number of “favorable mutations”, and that of “unfavorable mutations”. Favorable and unfavorable mutations were defined as IDH1, IDH2, NPM1, and CEBPA mutations, and ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and TP53 mutations, respectively, based on the previous reports (NEJM 2019, Blood 2022). The data were standardized and utilized for the linear discriminant analysis. Statistical analyses were performed using R version 4.2.3 software (R Foundation for Statistical Computation, Vienna, Austria). [Results] Total 158 patients were enrolled in our study. Among 48 patients who received Aza/Ven as a 1 st line therapy, NGS analyses were performed prior to treatment in 38 patients. Their median age was 70.5 (39-92) years old. AML with myelodysplasia-related changes was the major subtype (71%) and 37 percent of the cases had high risk cytogenetic abnormalities at diagnosis. After 1 st line Aza/Ven treatment, complete remission (CR)/ CR with incomplete blood count recovery (CRi) was achieved in 63% of the cases while the remaining 37% were non-CR. There was a trend that patients with NPM1 and IDH1/2 mutations achieved CR/CRi, while patients with RUNX1 mutation were not tended to achieve CR (Figure 1). Next, we performed linear discriminant analysis of treatment response using three pretreatment clinical factors. Linear discriminant analysis found a linear combination of prognostic factors that best separates two classes in the group of patients. Thirty-seven patients were employed to determine the discriminant function D, given by D = 0.638 × x − 0.76 × y + 0.68 × z + 0.145. Here x, y, and z represented the WT1 quantity in peripheral blood, the number of “favorable mutations”, and that of “unfavorable mutations” after standardization, respectively. Using this model, 81.1% of cases were correctly classified, and so were 75.7% of cases with leave-one-out cross validation method (Table 1). [Discussions] As previously reported (NEJM 2019, Blood adv 2020), IDH1/2 and NPM1 mutations tended to be sensitive to Aza/Ven in our cohort. However, it would be hard to predict the response of Aza/Ven by a single gene mutation because multiple gene mutations were co-existed in most of the cases (Figure 1) and clinical factors including tumor markers might also affect the outcome. Our mathematical model, involving gene mutations and WT1, could efficiently predict the response of Aza/Ven, which may support the selection of 1 st line treatment. In conclusion, our study revealed the genetic landscape of real-world Aza/Ven therapy and provided a potential prognostic model.

Delayed-Type Hypersensitivity: An Excellent Indicator of Anti-tumor Immunity With Wilms' Tumor 1 (WT1) Dendritic Cell Vaccine Therapy.

It is a well-known fact that anti-tumor immunity is a crucial long-term survival factor in cancer. Wilms' tumor 1 (WT1) dendritic cell vaccine therapy (WT1-DC) is an immuno-cell therapy that has been implemented against various cancers as a tumor-specific immunotherapy targeting the common cancer antigen WT1. Seven doses of WT1-DC vaccine were administered to six patients, three of whom had stage IV lung cancer with metastases and the other three had stage IV pancreatic cancer with metastases, all of whom were receiving chemotherapy and had similar physical conditions. Their immune response was assessed using delayed-type hypersensitivity (DTH) and immune profile status (IPS)such as blood neutrophil percentage, lymphocyte percentage, and neutrophil-to-lymphocyte (N/L) ratio. In lung cancer, DTH increased with repeated DC administration, and IPS improved with it, whereas in pancreatic cancer, DTH did not increase, and IPS worsened from the fifth inoculation. Fever in the 37° range was observed after DC administration in lung cancer, but not in pancreatic cancer. These results suggest that DTH and IPS are correlated in dynamics and that DTH is a good indicator of the state of anti-tumor immunity. Since IPS is a prognostic factor in advanced cancer, the magnitude of DTH due to WT1-DC inoculation is auseful indicator to estimate the patient's prognosis. Although DTH is an extremely simple test, its clinical significance has not been fully investigated. The present study demonstrates the importance of DTH in cancer treatment with WT1-DC.