Wilms Tumor 1 Research Articles

Generation of Monogenic Candidate Genes for Human Nephrotic Syndrome Using 3 Independent Approaches

IntroductionSteroid-resistant nephrotic syndrome (SRNS) is the second most common cause of chronic kidney disease during childhood. Identification of 63 monogenic human genes has delineated 12 distinct pathogenic pathways.MethodsHere, we generated 2 independent sets of nephrotic syndrome (NS) candidate genes to augment the discovery of additional monogenic causes based on whole-exome sequencing (WES) data from 1382 families with NS.ResultsWe first identified 63 known monogenic causes of NS in mice from public databases and scientific publications, and 12 of these genes overlapped with the 63 known human monogenic SRNS genes. Second, we used a set of 64 genes that are regulated by the transcription factor Wilms tumor 1 (WT1), which causes SRNS if mutated. Thirteen of these WT1-regulated genes overlapped with human or murine NS genes. Finally, we overlapped these lists of murine and WT1 candidate genes with our list of 120 candidate genes generated from WES in 1382 NS families, to identify novel candidate genes for monogenic human SRNS. Using this approach, we identified 7 overlapping genes, of which 3 genes were shared by all datasets, including SYNPO. We show that loss-of-function of SYNPO leads to decreased CDC42 activity and reduced podocyte migration rate, both of which are rescued by overexpression of wild-type complementary DNA (cDNA), but not by cDNA representing the patient mutation.ConclusionThus, we identified 3 novel candidate genes for human SRNS using 3 independent, nonoverlapping hypotheses, and generated functional evidence for SYNPO as a novel potential monogenic cause of NS.

Papers presented at the fall 2020 Pediatric Urologic Oncology Work Group of the Societies of Pediatric Urology meetingNeonatal Serum Electrolyte and Proteinuria Screening on 46,XY Ambiguous Genitalia Patients May Allow Early Diagnosis of Denys-Drash Syndrome: A Case Report

A term infant with prenatally noted ambiguous genitalia and nonpalpable gonads presented with life-threatening hyponatremia, hypertension, acidosis, and anuric renal failure requiring peritoneal dialysis at age 3 months.Sequencing confirmed 46, XY Denys-Drash syndrome (DDS) due to heterozygous Wilms tumor-1 exon 8 mutation encoding p.His445Arg. Renal US identified bilateral multifocal renal masses at age 8 months. Bilateral retroperitoneal nephrectomies found bilateral nephroblastomatosis without Wilms’ tumor avoiding chemotherapy, followed by bilateral laparoscopic orchiopexies. We suggest monthly screening of 46, XY DSD cases for DDS by evaluating for proteinuria and electrolyte disarray starting at diagnosis of DSD to prevent acute life-threatening renal failure presentation.

WT-1, BAALC, and ERG Expressions in Iranian Patients with Acute Myeloid Leukemia Pre- and Post-chemotherapy.

Purpose: Acute myeloid leukemia (AML) is the most prevalent acute leukemia in adults. It possesses different cytogenetic and molecular features. The expression of Wilms tumor-1 (WT1), brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) might be considered as prognostic factors in AML patients. The aim of this study was to determine the mRNA expressions of WT-1, BAALC and ERG genes in bone marrow of mononuclear cells and their effects on complete remission in the Iranian AML patients, pre- and post- chemotherapy. Methods: Forty AML patients with normal karyotype were evaluated. The mRNA gene expressions were measured with quantitative real-time PCR in bone marrow of mononuclear cells of AML patients at the baseline and after chemotherapy. The subtypes of AML and flow cytometry panel were also assessed. Complete remission (CR) after the treatment was addressed for all patients. Results: The mRNA expressions of WT-1, BAALC and ERG were significantly decreased after the treatment (p = 0.001, 0.017, 0.036). WT-1 mRNA expression was inversely correlated with CR after chemotherapy (P =0.024). There was also significant correlation between baseline expression of BAALC and CR (P =0.046). No significant correlation was observed between ERG and CR pre- and post- chemotherapy (P =0.464 and 0.781). There was also significant correlation between BAALC mRNA expression and CD34+ (P <0.001). Conclusion: The present study showed that WT-1 decreased significantly after standard chemotherapy which could have favorable effects on CR. Also, the high expression of BAALC could have a poor prognostic role in AML patients. The identification of these gene expressions can be an efficient approach in targeted therapy among AML patients.

The Genomic Landscape of Wilms' Tumor 1 (WT1) Mutant Acute Myeloid Leukemia

Mutations in tumor suppressor genes and oncogenes are both potentially therapeutically actionable in acute myeloid leukemia (AML). The Wilms' Tumor 1 (WT1) gene is located on 11p13 and encodes a zinc finger transcription factor which has been found to be overexpressed and mutated in AML. In normal development, WT1 is only expressed in a small subset of hematopoietic stem cells. While its overexpression suggests an oncogenic role, the invariable presence of mutations in the cysteine-histidine zinc finger domains indicates a tumor suppressor function, similar to that in WAGR syndrome/11p deletion syndrome in which it was first discovered. Like its unknown function in AML, the clinical significance and genetic associations of WT1 mutations have been also controversial. Although studies of WT1 mutations in AML have been conducted, the lack of solid clinical and molecular characterization of large WT1-mutant (WT1MT) AML cohort has hampered its definition. In this study, we took advantage of a compendia of genomic results from Cleveland Clinic and publicly available data of 2188 AML patients (primary (p)AML, n= 1636; secondary (s)AML, n= 433; therapy-related (t)AML, n= 119, excluding cases with acute promyelocytic leukemia, MLL-rearrangement, and core-binding factor AML). While several reports only focused on cytogenetic normal AML (CN-AML), which represented 61% of our cohort, we additionally included all other cytogenetic risk groups. In total, WT1 mutations were detected in 5% (114/2188) of patients. WT1 mutations were enriched in pAML (85%) compared to sAML (11%) and tAML (4%). Thirty-nine patients (13%) carried more than 1 WT1 mutation. WT1MT were younger [59 vs 64 years, P=0.0002] and more often females (55% vs 45%, P=0.03) as compared to WT1 wild type (WT1WT) patients. Univariate analyses of baseline parameters showed that WT1MT AML had a more proliferative phenotype with a higher WBC [15.1 vs 9.5 x109/L, P=0.03] and bone marrow blast percentages [73 vs 59%, P=0.002] and with lower platelet counts [44 vs 56 x109/L, P=0.008] compared to WT1WT cases. In the WT1MT cohort, 70% had a normal karyotype, with complex karyotype being significantly less frequent vsWT1WT patients [4 vs 16%, P=0.001]. The most common cytogenetic abnormalities in WT1MT patients included +8 (8%) followed by -9/del(9q) (3%) and -7/del(7q) (3%). Only 1 patient carried inv(3)/t(3;3) or -17/del(17p). In sum, no statistical differences in cytogenetics were found between WT1MTvsWT1WT AML patients. Next, identified mutational signatures of WT1MT patients. A panel of 44 myeloid genes and their hotspot configurations were selected according to their relevance in AML. In comparison to WT1WT AML patients, multivariate analyses showed that WT1MT patients had higher odds of biallelic CEBPA (12 vs 3%; P=0.009) and FLT3 internal tandem duplication mutations (FLT3ITD, 31 vs 16%; P=0.01) but lower odds of SRSF2 mutations (2 vs 9%, P=0.04). Since FLT3ITD has been previously described to be associated with WT1 mutations, we also focused on investigating whether mutations in the tyrosine kinase domain (TKD) were frequent in WT1MT as well. Although we found increased percentages of FLT3TKD (11%) among the WT1MT patients compared to WT1WT cohort (8%), this difference did not reach statistical significance. To uncover multifactor lesions (cytogenetic and/ or additional molecular lesions) of prognostic importance, we performed survival analyses. Although the combination of WT1 mutations and FLT3TKD shortened overall survival (OS) by 2-times in WT1MT patients vsWT1WT cases with FLT3TKD (23.7 vs 45.9 months), this result was not significant (P=0.1). In addition, the concurrent presence of other cytogenetic and molecular features didn't reveal significant impact on OS. In sum, using an adequately powered cohort, our study of the genomic landscape of WT1MT AML patients identified its genomic associations and their clinical and prognostic inferences. The application of advanced machine learning methods to large datasets of WT1MT AML patients might be crucial to capture the complex genomic interactions of WT1 gene in AML. Disclosures Carraway: BMS: Consultancy, Other: Research support, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Abbvie: Other: Independent Advisory Committe (IRC); Novartis: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau. Nazha:MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Incyte: Speakers Bureau; Jazz: Research Funding. Sekeres:Pfizer: Consultancy; BMS: Consultancy; Takeda/Millenium: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Donor-Derived T-Cells Specific for WT1 and PRAME in Combination with T-Cells Specific for Multiple Pathogens for Prevention of Relapse and Infection after Haemopoietic Stem Cell Transplant (HSCT) for Acute Myeloid Leukaemia (AML) or High-Risk Myelodysplasia (MDS) - (The INTACT Trial)

Introduction Disease relapse and infection cause significant morbidity and mortality after allogeneic HSCT for AML and MDS. Wilms' tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) are both commonly overexpressed in these conditions, and are attractive targets for immunotherapy. We have assessed the safety of a novel combination of tumour associated antigen (TAA) specific and multipathogen (MP) specific T cells administered prophylactically after HSCT in a phase 1 trial. Methods Patients were eligible for the study if WT1 or PRAME gene expression was elevated as determined by droplet digital PCR on diagnostic tumour samples. TAA and MP specific T cells were generated from stem cell donors by stimulating apheresis-derived mononuclear cells with autologous antigen presenting cells expressing tumour, viral or fungal antigens. T cells specific for CMV, EBV and Aspergillus antigens were produced separately and pooled in equal parts into a MP product. Patients received 1 infusion of MP specific T cells and up to 4 infusions of TAA specific T cells at 4-weekly intervals dosed at 2x107/m2, from 28 days post HSCT. Results Seven HSCT recipients have received a total of 26 T cell infusions to date. Median age was 49 years (range 26-67), disease AML (n=4) or high risk MDS (n=3), conditioning myeloablative (n=6) or reduced-intensity (n=1), donor source sibling (n=4) or matched unrelated (n=3). Median expression of WT1 on diagnostic bone marrow tissue was 1464 copies/104 copies of ABL (0-3870), PRAME 131 copies/104 copies of ABL (4-1670). Mean tumour antigen specificity in the TAA product was 2.2% of CD3+ cells for WT1 and 7.3% of CD4+ cells for PRAME. Mean total pathogen specificity in the MP product was approximately 15% (CMV=4.7% and EBV=5.4% of CD3+ cells, Aspergillus=5.3% of CD4+ cells). Patients received WT1 specific (n=3), PRAME specific (n=3) or both WT1 and PRAME specific T cells (n=1). All patients received MP specific T cells. No immediate infusion-related adverse events were reported. At the time of report, at a median of 375 days post-transplant (80-847), 5 out of 7 patients remain alive. Four patients remain in complete disease remission without graft versus host disease (GVHD). One patient did not proceed after 3 of 5 planned infusions after developing chronic lung GVHD but remains in disease remission. There have been 2 deaths (progressive disease and multiorgan failure). The patient with progressive disease had MDS with complex cytogenetics with evidence of persistent disease pre and post-HSCT, prior to T cell infusions. The patient with multiorgan failure had multiple post-transplant complications including bacterial sepsis, hepatic venoocclusive disease and grade 3 acute GVHD of the gut prior to infusion. Patients had low level viral reactivation (CMV n=3, EBV n=5, BKV n=3, HHV6 n=2), however none required treatment and there were no cases of viral tissue disease or EBV post-transplant lymphoproliferative disorder. There were no invasive fungal infections. Conclusion Prophylactic infusions of donor derived WT1/PRAME specific and multipathogen specific T cells post HSCT are well-tolerated and associated with low rates of infection and relapse in patients treated to date. Disclosures No relevant conflicts of interest to declare.

NTLA5001, a T Cell Product Candidate with CRISPR-Based Targeted Insertion of a High-Avidity, Natural, WT1-Specific TCR, Shows Efficacy in In Vivo Models of AML and ALL

Adoptive cell therapy using T cells expressing transgenic (tg) tumor antigen-targeting T cell receptors (TCRs) has become an attractive modality to treat hematological and solid cancers due to a broader array of accessible targets relative to CAR-T cell therapies. However, high-avidity TCRs specific for shared oncogenic antigens are difficult to identify. In addition, manufacturing of TCR-redirected T cells with single TCR specificity is desired to avoid mispairings and competition with endogenous chains, which can negatively impact T cell specificity and TCR expression levels. This can be achieved with CRISPR/Cas9-mediated replacement of the endogenous TCR α and β chains, by knocking out the TRAC and TRBC genes and inserting the tgTCR into the TRAC locus. While CRISPR/Cas9 genome editing has been demonstrated to be highly efficient, simultaneous edits in different loci could result in increased translocations, potentially impairing the quality and safety of the cell product. Moreover, existing cell engineering technology negatively impacts T cell quality and yield. Here, we focused on engineering T cells with specificity for Wilms' Tumor 1 (WT1), a transcription factor overexpressed by a wide range of hematological and solid tumors, that has both, restricted expression on healthy tissues and a strong correlation with oncogenesis. By applying rapid isolation technologies of WT1-specific T cells from healthy donors, we identified a lead TCR to the WT137-45 epitope, restricted to the common human leukocyte antigen, HLA-A*02:01. T cells expressing this tgTCR showed nM avidity and killed leukemia cell lines and primary acute myeloid leukemia (AML) blasts at low effector-to-target cell ratios. Epitope specificity evaluation by alanine scanning suggested that the minimal peptide recognition sequence for this TCR is restricted to WT1. Further, the lead TCR was able to activate CD8+ and CD4+ T cells, which may be beneficial for T cell persistence. By developing an improved T cell engineering process, we have achieved multiple sequential gene edits in primary human T cells, leading to knockout of the endogenous TCR with up to 99% efficiency and insertion of tgTCRs into 55-80% of the cells. This cell engineering process is scalable, adaptable to a closed system, and results in marked improvements in T cell expansion, yield, stem cell memory phenotype and T cell polyfunctionality, such as cytotoxicity, cytokine release and proliferation in response to WT1+ target cells. Additionally, the high viability profile of the process readily allows for sequential CRISPR/Cas9 gene knockout in T cells, leading to near-complete endogenous TCR removal while limiting TRAC/TRBC translocation to levels close to those found in untreated cells. T cells engineered to express the lead TCR using this process resulted in potent anti-tumor activity in vivo. Disseminated primary AML patient derived xenograft and acute lymphoblastic leukemia (ALL) cell line models were established by intravenous injection of the tumor cells in NSG or NOG mice. Animals were treated subsequently with WT1-specific or control T cells. Almost complete tumor growth inhibition in the blood and bone marrow was noted in the primary AML model. In the fast growing ALL model, WT1-T cells significantly reduced tumor burden and increased survival compared to control groups, which could be further boosted in human IL-15-expressing NOG mice vs. standard NOG mice. No signs of graph-versus-host disease (GvHD) were observed during the course of the study, which is consistent with removal of the endogenous TCR. NTLA-5001 is being advanced into clinical development for AML immunotherapy. Given the expression of WT1 in many solid tumors, engineered WT1 TCR-T cells are being further explored in those indications. Disclosures Liu: Intellia Therapeutics: Current Employment. Prodeus:Intellia Therapeutics: Current Employment. Becker:Intellia Therapeutics: Current Employment. Foisey:Intellia Therapeutics: Current Employment. Balwani:Intellia Therapeutics: Current Employment. Dutta:Intellia Therapeutics: Current Employment. Zhang:Intellia Therapeutics: Current Employment. Arredouani:Intellia Therapeutics: Current Employment. McKee:Intellia Therapeutics: Current Employment. Ciceri:Intellia Therapeutics: Membership on an entity's Board of Directors or advisory committees. Sepp-Lorenzino:Intellia Therapeutics: Current Employment. Bonini:Kiadis: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; Molmed: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Intellia Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties, Research Funding. Schultes:Intellia Therapeutics: Current Employment, Current equity holder in publicly-traded company.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that both affect the epigenetic regulation of DNA methylation. Although the proteins antagonistically regulate the epigenetic mark of 5-methylcytosine (5-mC), where DNMT3A catalyzes addition of 5-mC while TET2 oxidizes 5-mC as a first step in DNA demethylation, mutations in both genes appear in a similar spectrum of human hematopoietic malignancies. Mutations in both genes often co-occur with activating mutations in oncogenic tyrosine kinases (OTKs) such as FLT3ITD, BCR-ABL1, JAK2V617F, MPLW515L or mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Moreover, while the mutations exert divergent effect on primitive hematopoietic progenitor cells, they lead to similar disease phenotypes, suggesting the roles of these mutations in hematopoietic malignancies may relate to mechanisms outside of DNA methylation. OTK-positive malignant cells accumulate high numbers of spontaneous and drug-induced DNA double-strand breaks (DSBs) in comparison to normal cells, but they manage to survive because of their enhanced/altered ability to repair these breaks. DSBs, the most lethal DNA lesions, are repaired by two major mechanisms, BRCA1/2-dependent homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (D-NHEJ). Both HR and D-NHEJ repair DSBs in proliferating cells, while D-NHEJ plays a major role in quiescent cells. PARP1 -dependent alternative NHEJ (Alt-NHEJ) serves as back-up in both proliferating and quiescent cells. The existence of these redundant pathways creates the opportunity to employ a phenomenon called "synthetic lethality", which was originally applied to eliminate cancer cells with mutations in BRCA1 and BRCA2 by PARP inhibitor (PARPi). Our previous report [M. Nieborowska-Skorska et al., Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells. J Clin Invest 127, 2392-2406 (2017)] suggested that certain leukemias are sensitive to PARPi-triggered synthetic lethality. Here we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing OTKs. Malignant TET2-deficient cells display downregulation of BRCA1 and LIG4 resulting in reduced activity of HR and D-NHEJ, respectively, and rely on Alt-NHEJ to protect them from the toxic effects of replication stress and drug-induced DSBs. Conversely, DNMT3A-deficient cells favor HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells are sensitive to PARPi treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity and/or TET2 - Wilms tumor 1 (WT1) binding ability were responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Our findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Tallman:Abbvie: Research Funding; Cellerant: Research Funding; Orsenix: Research Funding; ADC Therapeutics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Rafael: Research Funding; Amgen: Research Funding; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents &amp; Royalties. Martinelli:Roche: Consultancy; Pfizer: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Daichii Sankyo: Consultancy, Research Funding; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; AbbVie: Consultancy, Research Funding. Vassiliou:Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.

Frequency and Stimulation of NPM1-Specific Immune Responses By Anti-PD1 Antibodies in NPM1-Mutated AML

Nucelophosmin1 (NPM1) is one of the most commonly mutated genes in AML, represents a distinct entity according to the WHO and is commonly associated with a favorable prognosis. We described specific immune responses against immunogenic epitopes derived from the mutational region of NPM1 in AML and specific immune responses against leukemic progenitor and stem cells (LPC/LSC). Immune responses play an increasing role in AML treatment options. However, the role of Immuncheckpoint inhibition is still controversially discussed in AML. In this work, we investigated NPM1 specific immune responses but also responses against other leukemia associated antigens (LAA) like PRAME (P300), Wilms' Tumor 1 (WT1) and RHAMM (R3) in an extended cohort. We investigated these immune responses in LSC/LPC using colony-forming immunoassays (CFI) and ELISpots. We examine whether immuncheckpoint inhibition using the anti-programmed death 1 (anti-PD-1) antibody might increase immune responses against stem cell like cells, comparing NPM1-mutated (NPM1mut) to NPM1 wildtype (NPM1wt) patients. In AML NPM1mut patients, specific immune responses of cytotoxic T cells against the epitope NPM1 showed a mean reduction of colonies in CFI of 27%, for P300 of 38%, for WT1 of 32% and for R3 of 42%. NPM1wt patients showed a mean reduction of colonies in CFI of for P300 of 28%, for WT1 of 28% and for R3 of 41%. Results are comparable, for NPM1mut and NPM1wt patients, there were immune responses seen in all epitopes. When adding the anti-PD-1 antibody to CFI, in NPM1mut patients the mean additional inhibition using NPM1 as target was 47%, and for the other epitopes: P300 23%, WT1 27% and R3 26%. For NPM1WT patients, the additional inhibition with the addition of anti-PD-1 to CFI was for P300 25%, for WT1 34% and for R3 23%. For the NPM1 epitope, 8 of 10 NPM1mut patients showed an immune response and 3/10 showed reduction in CFI of &amp;gt; 50%. The NPM1 epitope displayed an additional reduction with anit-PD-1 in all 10 NPM1mut Patients and in 6 of 10 a reduction of &amp;gt; 50%. Thus, especially the NPM1 specific immune responses are strong in NPM1mut patients by adding anti-PD-1. Taken together, the anti-PD-1 antibody increases specific T cell responses of LAA-stimulated CTL and the cytotoxic effect of T cells against LPC/LSC. The effect was strongest in NPM1mut patients against the immunogenic epitope derived from the mutational region of NPM1 and even stronger when adding anti-PD-1. These data suggest there could be a NPM1 mutation directed immunotherapeutic approach using LAA-directed vaccination strategies in NPM1-mutated AML and combination with anti-PD-1 antibodies might open new application possibilities. Disclosures Greiner: BMS: Research Funding. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding.

MiR-199a-5p stimulates ovarian granulosa cell apoptosis in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and one of the most common causes of infertility in women. PCOS patients have been found with dysregulated miRNA, which is indicative of their roles as noninvasive biomarkers and novel therapeutic targets in PCOS. Herein, this study sets out to explore the mechanism of action of miR-199a-5p in PCOS in relation to the janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway via Wilms' tumor 1 (WT1) regulation in a rat model of PCOS. The expression of miR-199a-5p was highly expressed in ovarian cortical tissues and serum of PCOS patients, as examined by RT-qPCR. Ovarian granulosa cells (GCs) were harvested from PCOS rat model, followed by subsequent purification. Gain- and loss-of-function experiments of miR-199a-5p were performed to determine its functions in PCOS. Cell viability, cell apoptosis and serum hormone levels were assessed, the results of which showed that downregulation of miR-199a-5p contributed to the promotion of GC viability and inhibition of apoptosis, while simultaneously inducing the elevation of serum E2 level and reduction of serum AMH, PG, LH and FSH levels in the PCOS rat model. WT1 was identified as a target gene of miR-199a-5p by dual-luciferase reporter gene assay, and inhibition of miR-199a-5p resulted in the activation of WT1-mediated JAK/STAT3 pathway. The activated JAK/STAT3 pathway suppressed the development of PCOS by miR-199a-5p, indicating a mechanism by which miR-199a-5p could potentially prevent PCOS through the WT1-mediated JAK/STAT3 pathway.

Wilms' Tumor 1 (WT1): The Vaccine for Cancer.

Vaccines have been used to fight and protect against infectious diseases for centuries. With the emergence of immunotherapy in cancer treatment, researchers began investigating vaccines that could be used against cancer, especially against tumors that are resistant to conservative chemotherapy, surgery, and radiotherapy. The Wilms' tumor 1 (WT1) protein is immunogenic, has been detected in almost all types of malignancies, and has played a significant role in prognosis and disease monitoring. In this article, we review recent developments in the treatment of various types of cancers with the WT1 cancer vaccine; we also discuss theoretic considerations of various therapeutic approaches, which were based on preclinical and clinical data.

Metastases to the Breast from Extramammary Nonhematological Malignancies: Case Series.

ObjectiveThis article aims to provide a better understanding of ultrasonography and immunohistochemistry of secondary nonhematological tumors of breast.MethodsThe study reviewed the ultrasound findings and immunohistochemical features of nonhematological metastatic breast tumor cases found in patients of West China Hospital, Sichuan University from 2007 to 2019. Each case was categorized as secondary breast malignancy using histopathological results.ResultsFourteen cases were identified from West China Hospital database. Ten cases originated in the lung, 2 cases in the stomach, 1 case in the ovary and 1 case of neuroendocrine carcinomas. Fourteen masses were evaluated. Ultrasound findings showed that tumors were hypoechoic (14/14), irregular (13/14), indistinct margin (13/14), along a long axis parallel to the skin (11/14), lacked vascularity via color doppler flow imaging (9/14). Eight cases showed no posterior features. Calcification was found in 1 case of lung adenocarcinoma that had metastasized to the breast. Abnormal axillary lymph nodes were detected in 5 cases. Immunohistochemical analysis showed that estrogen receptor (ER) and progesterone receptor (PR) were both negative in 11 cases, including gastric and lung cancer metastasis. One case of ovarian metastasis was positive for ER and negative for PR. Six patients were positive for cytokeratin 7 (CK7) and negative for cytokeratin 20 (CK20), including lung and ovarian carcinoma metastasis. Thyroid transcription factor-1 (TTF-1) was positive in 9 of 10 pulmonary carcinoma metastases. The patient of ovarian metastasis was positive for Wilms’ tumour 1 (WT-1) and carbohydrate antigen 125 (CA125). Two cases from gastric metastasis were positive for caudal-type homeobox 2 (CDX2).ConclusionAlthough breast ultrasound is not useful in distinguishing metastases from primary breast cancer, it is helpful in diagnosing breast lesions as oncological diseases and provide evidence for further examination of patients. Immunohistochemistry plays an important role in distinguishing secondary breast cancer from primary, especially in patients without tumor history.

Novel WT1 Target Genes: IL-2, IL-2RB, and IL-2RG Discovered during WT1 Silencing Using Lentiviral-Based RNAi in Myeloid Leukemia Cells.

Wilms' tumor 1 (WT1) is a transcription factor which plays a major role in cell proliferation, differentiation, survival, and apoptosis. WT1 was first identified as a tumor suppressor gene in Wilms' tumor. However, overexpression of WT1 has been detected in several types of malignancy including some types of leukemia. To investigate the molecular mechanism underlying WT1-mediated leukemogenesis, lentiviral-based siRNA was employed as a tool to suppress WT1 expression in the myeloid leukemia cell line, K562. Successfully, both WT1 RNA and protein levels were downregulated in the leukemia cells. The silencing of WT1 resulted in significant growth inhibition in WT1-siRNA-treated cells for 40 ± 7.0%, 44 ± 9.5%, and 88 ± 9.1% at 48, 72, and 96 hours posttransduction as compared with the control cells, respectively. By using apoptosis detection assays (caspase-3/7 activity and Annexin V-FITC/PI assays), WT1 silencing induced a higher degree of early and late apoptosis in siRNA-treated K562 as compared with the control cells. Interestingly, the expression of survival signaling genes, IL-2, IL-2RB, and IL-2RG, was also suppressed after WT1-siRNA treatment. In addition, the WT1 silencing also inhibited the S phase of the cell cycle and induced cell death. Our results indicated that WT1 silencing by siRNA can suppress cellular proliferation, induce apoptosis, and reduce S phase fraction of K562 cells. Moreover, transcriptional modulation of IL-2, IL-2RB, and IL2-2RG expression by WT1 was likely involved in this phenotypic change. Overall, this study confirmed the oncogenic role of WT1 in myeloid leukemia and discovered the new target genes of WT1 which are likely involved in WT1-mediated leukemogenesis.

Wilms' tumor 1 antigen immunoreactivity in epithelial ovarian cancer - diagnostic and prognostic value.

Ovarian cancer is a heterogeneous disease, with a number of different histological subtypes with various responses to treatment. Wilms' tumor 1 (WT1) immunoreactivity is used to distinguish between OC's various subtypes. However, little is known about the protein's role as a prognostic factor. Thus, the main aim of our study was to evaluate the relationship between WT1 expression and patient overall survival (OS) and lymph node metastases. Study group consisted of 164 women aged 22-84, diagnosed with epithelial ovarian cancer (EOC). WT1 expression in histological slides was assessed by immunohistochemistry. Serous tumors were the most common subtype among EOC (n = 126; 76.8%), followed by endometrioid (n = 20; 12.2%), clear-cell (n = 14; 8.5%) and mucinous cancer (n = 4; 2.4%). Of all serous EOC, WT1-positive tumors accounted for 75.6% of cases and this number was significantly higher than in other histological subtypes (p < 0.0001). Patients with lymph node metastases were more likely to have WT1-positive than WT1-negative tumors (p = 0.006). There was no significant correlation between WT1 immunoreactivity and OS across the whole study group of EOC patients (p = 0.6); however, in the group of non-serous (mucinous, endometrioid and clear-cell) EOC subjects, WT1 immunoreactivity was associated with shorter OS (p = 0.046). WT1 immunoreactivity may be helpful in differentiating primary epithelial serous carcinomas from non-serous ovarian cancers; however, its prognostic role in EOC is rather uncertain.

Enhanced immune reaction resulting from co-vaccination of WT1 helper peptide assessed on PET-CT.

It has become evident that positron emission tomography/computed tomography (PET-CT) using 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) (FDG PET-CT) can detect anti-tumor immune response induced by various immunotherapies. To evaluate whether FDG PET-CT could detect anti-cancer immune response caused by cancer vaccine therapy, we performed a retrospective analysis of FDG PET-CT imaging of patients who were treated with Wilms Tumor 1 (WT1) vaccine therapy in Osaka University during July 2008 and June 2018. Increased FDG uptakes were detected in WT1-vaccinated skin and their draining lymph nodes during the repeated vaccination. While the FDG uptakes seemed to decrease with time after the cessation of WT1 peptide vaccinations, persistence of FDG uptakes for years in WT1-vaccinated skin were also observed in 2 cases who showed good clinical course. Moreover, the FDG uptakes of patients treated with the combination vaccine of WT1 specific cytotoxic T cell (CTL) and helper peptides were significantly stronger than of those treated with the WT1 CTL peptide alone. Since it is evident that the combination vaccine can induce a more robust anti-tumor immunity than can CTL peptide vaccine alone, the FDG uptakes in WT1-vaccinated skin might reflect the degree of immune response. These results suggest that PET-CT might be a good tool for prediction of anti-tumor immune response induced by WT1 vaccine therapy. Larger scale prospective studies therefore seem to be warranted.

Clinical Grade Production of Wilms' Tumor-1 Loaded Cord Blood-Derived Dendritic Cells to Prevent Relapse in Pediatric AML After Cord Blood Transplantation.

Hematopoietic cell transplantation (HCT) is a last resort, potentially curative treatment option for pediatric patients with refractory acute myeloid leukemia (AML). Cord blood transplantation (CBT) results in less relapses and less graft-versus-host disease when compared to other sources. Nevertheless, still more than half of the children die from relapses. We therefore designed a strategy to prevent relapses by inducing anti-AML immunity after CBT, using a CB-derived dendritic cell (CBDC) vaccine generated from CD34+ CB cells from the same graft. We here describe the optimization and validation of good manufacturing practice (GMP)-grade production of the CBDC vaccine. We show the feasibility of expanding low amounts of CD34+ cells in a closed bag system to sufficient DCs per patient for at least three rounds of vaccinations. The CBDCs showed upregulated costimulatory molecules after maturation and showed enhanced CCR7-dependent migration toward CCL19 in a trans-well migrations assay. CBDCs expressed Wilms’ tumor 1 (WT1) protein after electroporation with WT1-mRNA, but were not as potent as CBDCs loaded with synthetic long peptides (peptivator). The WT1-peptivator loaded CBDCs were able to stimulate T-cells both in a mixed lymphocyte reaction as well as in an antigen-specific (autologous) setting. The autologous stimulated T-cells lysed not only the WT1+ cell line, but most importantly, also primary pediatric AML cells. Altogether, we provide a GMP-protocol of a highly mature CBDC vaccine, loaded with WT1 peptivator and able to stimulate autologous T-cells in an antigen-specific manner. Finally, these T-cells lysed primary pediatric AML demonstrating the competence of the CBDC vaccine strategy.

Expansion of the renal capsular stroma, ureteric bud branching defects and cryptorchidism in mice with Wilms tumor 1 gene deletion in the stromal compartment of the developing kidney.

Development of the mammalian kidney is orchestrated by reciprocal interactions of stromal and nephrogenic mesenchymal cells with the ureteric bud epithelium. Previous work showed that the transcription factor Wilms tumor 1 (WT1) acts in the nephrogenic lineage to maintain precursor cells, to drive the epithelial transition of aggregating precursors into a renal vesicle and to specify and maintain the podocyte fate. However, WT1 is expressed not only in the nephrogenic lineage but also transiently in stromal progenitors in the renal cortex. Here we report that specific deletion of Wt1 in the stromal lineage using the Foxd1cre driver line results at birth in cryptorchidism and hypoplastic kidneys that harbour fewer and enlarged ureteric bud tips and display an expansion of capsular stroma into the cortical region. In vivo and ex vivo analysis at earlier stages revealed that stromal loss of Wt1 reduces stromal proliferation, and delays and alters branching morphogenesis, resulting in a variant architecture of the collecting duct tree with an increase of single at the expense of bifurcated ureteric bud tips. Molecular analysis identified a transient reduction of Aldh1a2 expression and of retinoic acid signalling activity in stromal progenitors, and of Ret in ureteric bud tips. Administration of retinoic acid partly rescued the branching defects of mutant kidneys in culture. We propose that WT1 maintains retinoic acid signalling in the cortical stroma, which, in turn, assures proper levels and dynamics of Ret expression in the ureteric bud tips, and thus normal ramification of the ureteric tree. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Wilms’ tumor 1 (WT1) promotes ovarian cancer progression by regulating E-cadherin and ERK1/2 signaling

ABSTRACT Wilms’ tumor 1 (WT1) is reported to play an important role in tumor invasion and metastasis, two hallmarks of ovarian cancer (OC) that influence treatment efficacy and prognosis. However, the specific roles and underlying mechanisms of WT1 in OC have not been fully understood. Here, we investigated the potential function and signaling pathways of WT1 in OC cells. We showed that WT1 was significantly upregulated in human OC tissues and closely associated with OC type, grade and FIGO stage. In cultured cells and xenograft mouse models, WT1 depletion significantly inhibited cell migration and invasion, reversed epithelial–mesenchymal transition (EMT), and prevented metastasis of OC cells. We further demonstrated that WT1 inhibited E-cadherin expression via targeting E-cadherin gene promoter by chromatin immunoprecipitation and luciferase reporter assay. Moreover, ERK1/2 activation was suppressed upon WT1 silencing. Inhibiting ERK1/2 phosphorylation increased E-cadherin expression and suppressed WT1-induced OC cell migration and invasion. Taken together, our study reveals WT1 exerts a tumor-promoting role in OC, enhancing EMT through negative modulation of E-cadherin expression via ERK1/2 signaling. WT1 may represent a novel therapeutic target that may improve the prognosis of OC.

MicroRNA-466o-3p mediates β-catenin-induced podocyte injury by targeting Wilms tumor 1.

Podocytes are highly specialized cells that play an essential role in maintaining the integrity and function of the glomerular filtration barrier. Wilms tumor 1 (WT1) and β-catenin are two master regulators that play opposing roles in podocyte biology and mutually antagonize each other. However, exactly how β-catenin inhibits WT1 remains incompletely understood. In this study, we demonstrated the role of miR-466o-3p in mediating β-catenin-triggered podocyte injury by targeting WT1. The expression of miR-466o-3p was upregulated in cultured podocytes after β-catenin activation and in glomerular podocytes in adriamycin (ADR) nephropathy, remnant kidney after 5/6 renal ablation, and diabetic kidney disease. Bioinformatics analysis and luciferase reporter assay confirmed that miR-466o-3p directly targeted WT1 mRNA. Furthermore, overexpression of miR-466o-3p downregulated WT1 protein and promoted podocyte injury in vitro. Conversely, inhibition of miR-466o-3p alleviated β-catenin-induced podocyte dysfunction. In mouse model of ADR nephropathy, overexpression of miR-466o-3p inhibited WT1, aggravated podocytes injury and deteriorated proteinuria. In contrast, inhibition of renal miR-466o-3p by antagomiR, either prior to or after ADR injection, substantially restored WT1, alleviated podocytes injury and reduced renal fibrosis. These studies reveal a critical role for miR-466o-3p, a novel microRNA that has not been characterized previously, in mediating β-catenin-triggered WT1 inhibition. Our findings also uncover a new pathogenic mechanism by which β-catenin promotes podocyte injury and proteinuria in glomerular diseases.

Wilm's Tumor 1-guided preemptive treatment with hypomethylating agents for molecular relapse of AML and MDS after allogeneic transplantation.

Hypomethylating agents (HMA) for relapsed acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) after allogeneic transplantation (allo-SCT) are most effective when used at the stage of molecular relapse. As Wilm's Tumor 1 (WT1)- expression has proven to serve as broadly applicable, sensitive and specific minimal residual disease (MRD) marker, we measured WT1-expression in 35 AML and MDS patients using a standardized assay for the guidance of therapy with HMA and donor lymphocyte infusions (DLI). Molecular relapse was detected in median 168 days post-transplant prompting therapy with a median of six HMA cycles and at least one DLI (n = 22, 63%). Hereby, 13 patients (37%) achieved major response (=MRD- complete remission [CR]), and 7 patients (20%) achieved minor response (=MRD+ CR), whereas 15 patients (43%) progressed into hematologic relapse. Two-year overall survival (OS) rate was 35% including 11 patients (31%) with ongoing MRD- remission for a median of 21 months. Patients with the major response after six cycles had significantly better OS suggesting that those not achieving MRD negativity after six cycles are candidates for alternative therapies. Combining MRD-monitoring of WT1-expression and preemptive therapy with HMA and DLI appears as a practicable and efficient approach for imminent relapse after allo-SCT.

Podocyte Involvement in Renal Thrombotic Microangiopathy: A Clinicopathological Study

Background: The current study aimed to evaluate the associations between podocyte injury and clinicopathological features in renal thrombotic microangiopathy (TMA) based on a Chinese cohort, which might be underscored in this disease. Methods: The clinical, laboratory, and renal histopathological data of patients with renal biopsy-proven TMA from 2000 to 2015 in our institute were collected. Foot process effacement (FPE) was quantified by foot process width (FPW) by electron microscopy. Podocytes in the renal specimens were also detected by stainings for podocyte-specific markers, including Wilms tumor 1 (WT-1), synaptopodin, and podocalyxin. The associations between FPW and clinico-histopathological data were further analyzed. A composite end-point was defined by all-cause death or end-stage renal disease to address the predictive value of FPW. Results: Sixty-three patients with renal biopsy-proven TMA were enrolled. The FPW of renal TMA patients was 1,090 ± 637 nm (range, 572–4,748 nm), which was significantly higher than the normal range in our center (p = 0.005). By immunohistochemistry and immunofluorescence assays, we found decreased expressions of synaptopodin, podocalyxin, and WT-1 and continued stainings of WT-1 in some podocytes without detectable synaptopodin stainings in the areas of sclerotic tufts and cellular crescents. The FPW value was correlated with the serum albumin concentration (r_s = −0.281, p = 0.026), proteinuria amount (r_s = 0.255, p = 0.047), serum creatinine levels (r_s = 0.339, p = 0.007), and eGFR (r_s = −0.335, p = 0.007). According to ROC curve analysis, the optimal cutoff level of FPW for predicting the composite end-point was 869 nm. In patients with FPW ≥ 869 nm, FPW levels were further correlated with the severity of mesangiolysis (r_s = 0.351, p = 0.033) and glomerulosclerosis (r_s = 0.369, p = 0.025) in pathological evaluations. Patients without clinical remission also had higher FPW than those with remission (1,240 ± 793 vs. 925 ± 344 nm, p = 0.013). The multivariate Cox hazard model showed that FPW ≥ 869 nm was an independent risk factor for the composite end-point (hazard ratio: 3.64, 95% CI: 1.37–9.66, p = 0.009). Conclusion: The podocyte injury was prevalent and the FPW levels were closely associated with clinicopathological features, especially prognosis, in renal TMA patients.