Wilms Tumor 1 Research Articles

Immunofluorescence staining of WT-1/Podocalyxin on Mouse kidney sections.

In glomerular diseases, podocytes are one type of cells involved in dysfunction of glomerular filtration. In these conditions, podocyte proteins expression change. Therefore, immunofluorescent staining of podocytes can be performed to visualize podocyte localization of proteins of interest. In this protocol, we detail a method to stain podocytes with a specific marker WT-1 (Wilms Tumor-1) for "in situ" podocyte analysis by immunofluorescent microscopy, more informative technique than other techniques (as Western blot).

Wilm's tumor-1 (WT1) rs16754 polymorphism

Wilm's tumor-1 (WT1) rs16754 polymorphism

The clinical significance of the alternative Wilms tumor gene overexpression-hypermethylation signature in acute myeloid leukemia.

Wilms tumor 1 (WT1) gene is overexpressed in numerous cancers, including acute myeloid leukemia (AML). The alternative WT1 gene (AWT1) is generated from alternative transcription start site in the WT1 first intron and encodes an N-terminal-truncated protein lacking the repressor domain. Although WT1 overexpression is a common feature in AML, the expression levels of the AWT1 and its underlying epigenetic alterations, as well as their clinical relevance in AML remain unknown. Quantitative assessment of AWT1 gene transcripts was performed using real-time polymerase chain reaction (PCR). Bisulfite PCR followed by pyrosequencing was done to determine the methylation status of the AWT1 promoter. The bone marrow samples were collected at diagnosis and after completion of induction chemotherapy from 80 newly diagnosed AML patients. Forty non-malignant BM samples were recruited as controls. The AWT1 was significantly overexpressed in AML patients. Robust hypermethylation of the AWT1 promoter was found to be a highly specific and sensitive marker for AML (p < 0.001). Significant positive correlations between the AWT1 expression and methylation levels with BM blast counts at both initial diagnosis and after induction therapy were observed (p < 0.001). AWT1 overexpression at the initial diagnosis of AML was found to be an independent negative factor for complete remission response after induction therapy (p = 0.014). The AWT1 gene overexpression-hypermethylation signature is a characteristic marker that positively correlates with the leukemic burden in AML. AWT1 overexpression at AML diagnosis is an independent negative predictor for CR after induction chemotherapy.

WT1 mRNA Expression in the Peripheral Blood of Patients with MDS Yields Additional Prognostic Information within the IPSS-R Risk Categories “Very Low”, “Low” and “Intermediate”

Introduction:The revised version of the International Prognostic Scoring System, (IPSS-R), is an accepted standard for assessing the prognosis of patients with MDS. Its usefulness may be further improved by integrating molecular findings. However, such efforts are impeded by limited access to molecular diagnostics, lack of standardized methodology, and a relatively low frequency of individual gene mutations in MDS. The Wilms' Tumor-1 (WT1) gene is overexpressed on mRNA level in the peripheral blood (PB) in about 50% of patients with MDS. The aim of this analysis was to determine whether PB WT1 expression status yields additional prognostic information.Methods:For this purpose, PB WT1 mRNA expression was measured in 91 newly diagnosed patients with MDS (WHO: MDS del5q, n=7; RARS, n=1; RCUD, n=4; RCMD, n=37; RAEB-I, n=16; RAEB-II, n=23; MDS/MPN unclassifiable, n=3 / IPSS-R: very low risk, n=3; low risk, n=28; intermediate risk, n=27; high risk, n=13; very high risk, n=20), using the Ipsogen® WT1 ProfileQuant® Kit. This standardized, commercially available assay uses a validated cut-off level of 50 WT1 copies/104ABL copies for discrimination between normal and overexpression of WT1 in PB. MDS patients in our study cohort were stratified accordingly (normal WT1 expression with <50 WT1 copies versus overexpression with >50 WT1 copies). WT1 expression status was correlated with clinical parameters and outcome.Results:Overall, 53 MDS patients (58%) showed WT1 overexpression, which correlated significantly with WHO 2008 disease category and IPSS-R risk groups, as indicated by both the absolute WT1 levels (correlation with WHO 2008 type, p=0.0028, and IPSS-R, p=0.0075) and the frequency of WT1-overexpressing patients within the respective MDS subgroup (correlation with WHO 2008 type, p=0.0029, and IPSS-R, p=0.0027). Regarding the entire cohort, patients with elevated WT1 expression had a significantly lower progression-free survival (PFS) and overall survival (OS) compared to those with normal WT1 expression (p<0.0001 and p=0.0306). Furthermore, within the IPSS-R risk groups ‘very low’, ‘low’ and ‘intermediate’, PFS differed significantly between patients showing normal vs. elevated WT1 expression status (IPSS-R very low/low: median PFS 30.1 months vs. not reached, for WT1 high vs normal, respectively, p=0.0127; IPSS-R intermediate: median PFS 14.4 months vs. 59.5 months, for WT1 high vs. normal, respectively p=0.0240). These differences in PFS retained their prognostic significance in multivariate analysis after adjusting for IPSS-R (HR 0.306; 95% CI 0,156-0,598, p=0.001). However, they did not translate into a difference in overall survival, which was probably due to a relevant number of patients proceeding to allogeneic stem cell transplantation. Given the large proportion of patients displaying WT1 overexpression in the IPSS-R high and very high risk groups, it was not surprising that no significant prognostic subdivision by WT1 expression level was seen in these risk categories.Conclusion:Our results show that PB WT1 expression offers additional prognostic information in patients belonging to the IPSS-R risk groups ‘very low’, ‘low’ and ‘intermediate’. Assessment of WT1 expression status at diagnosis is a relatively time and cost efficient method that can be performed without patient discomfort and may help to identify MDS IPSS-R low and intermediate patients at risk for early progression. DisclosuresRautenberg:Celgene: Honoraria. Germing:Janssen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kobbe:Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel Support, Research Funding. Schroeder:Celgene: Consultancy, Honoraria, Research Funding.

Overexpression mRNA of HMGA2 and WT1 Genes in Whole Blood Samples As Potential Diagnostic Markers in MPN

BackgroundHigh-mobility group AT-hook protein 2 (HMGA2) gene codes for a non-histone protein which expressed during embryogenesis and early hematopoiesis but suppressed in differentiated cells. Overexpression of HMGA2 as well as transcription factor Wilms' tumor 1 (WT1) gene has been found in a variety of human cancers, but only a few reports has included myeloproliferative neoplasms (MPNs) (Guglielmelli P. et al., 2007; Chen C-C. et al., 2017; Tasdemir S. et al, 2015). Furthermore, there were no any data about co-expression of mRNA both genes in blood in patients with MPNs.Aims: evaluate of the diagnostic potential of the WT1 and HMGA2 mRNA level in MPN patients.MethodsIn our retrospective study was analysed 115 archived frozen peripheral blood samples with RNAse inhibitor from 62 MPN patients: 29 with polycythemia vera (PV), 16 with essential thrombocythemia (ET), and 17 with myelofibrosis (MF) were included. Samples of 28 healthy volunteers were tested as control group. Total RNA was isolated using “Ribo-zol-D” (Aplisens) and then were transcribed using “Reverta-L” (Aplisens). Synthesized cDNA was analysed by real-time PCR to detect of WT1 and HMGA2 expression levels on the CFX96 (Bio-Rad) housekeeping kits using. The results were calculated utilizing the delta Ct method in the software package of “R”. The threshold cycles (Ct) WT1 and HMGA2 genes and reference gene ABL determined using Cy0 method. Results are presented as median and range (25% and 75%). Comparisons between the patient and control group were made using the Mann-Whitney U test. Statistical analysis was performed using Statistica 12.0 software.ResultsThe level of HMGA2 mRNA in five samples of venous blood in PV patients and 12 samples in MF patients was higher than in healthy subjects (Figure 1). Expression of WT1 mRNA (≥ 0.01%) was observed only in samples among patients with MF (in 19 of 28). In 12 blood samples of patients with MF, transcripts of both genes were detected together (Table 1). The increased level of HMGA2 mRNA expression was associated with a higher level of erythrocytosis in patients with PV (p<0.05). The co-expression of HMGA2 mRNA and WT1 mRNA in patients with MF was associated with a low level of white and red blood cell (p<0.05). Interestingly that in MF patients the most pronounced thrombocytopenia was observed if only WT1 mRNA was detected. In all cases of repeated testing of patients, the growth of HMGA2 or WT1 mRNA level was accompanied by decline of the cellular blood composition. The level of HMGA2 mRNA and WT1 mRNA in our study did not depend on the driver mutations presence or their allele burden. It should be noted that 4 out of 12 patients with MF died within 5 months after the first detection of WT1 mRNA and one patient died more after 15 months. At that time only one MF patient without WT1 or HGMA2 transcripts died during of 20 months' observation.Summary and ConclusionsExpression of the WT1 and HMGA2 genes is involved in the pathogenesis and progression of the disease and can be used as diagnostic markers. The question of the independent prognostic significance of these transcripts in blood samples for various phenotypes of MPN is promising for further study. DisclosuresNo relevant conflicts of interest to declare.

Phase II Trial of a Peptide Vaccine, Ocv-501 in Elderly Patients with Acute Myeloid Leukemia

Introduction: In recent decades, a considerable number of tumor antigens have been characterized, including the leukemia-associated antigen, Wilm's tumor 1 (WT1). WT1 is found to be expressed at low levels in various normal tissues such as gonads, kidney and the hematopoietic system. However, it is highly overexpressed in various hematological malignancies and solid tumors, including in the majority of acute myeloid leukemia (AML) cases. OCV-501 is a human leukocyte antigen (HLA) class II-restricted helper peptide derived from the WT1 protein. This Phase II trial was conducted to evaluate the efficacy and safety of OCV-501 in elderly patients with AML (ClinicalTrials.gov identifier: NCT01961882).Methods: This multicenter, randomized, double-blind, placebo-controlled trial was conducted in Japan, the Republic of Korea and Taiwan between October, 2013 to November, 2017. The key eligibility criteria for this trial were patients with AML, aged 60 years or older, who achieved first complete remission within one or two courses of standard induction therapy, completed more than one course of standard consolidation therapy, and not scheduled for hematopoietic stem cell transplantation. Patients were assigned to receive either OCV-501 monotherapy or placebo. Subcutaneous administration of OCV-501 3mg or placebo was given once weekly up to the eighth administration, and once biweekly from the ninth administration onwards up to a duration of 2 years, followed by post-treatment observation period. The primary endpoint was disease-free survival (DFS) that was defined as the time from randomization to AML relapse or death from any cause, whichever came first, within the observation period. Secondary endpoints were overall survival (OS), quality of life (QOL; The European Organization for Research and Treatment of cancer [EORTC] QLQ-C30), and performance status (Eastern Cooperative Oncology Group performance status [ECOG PS]). Safety, including any occurrence of adverse events (AEs) was evaluated.Results: A total of 133 patients who had achieved first remission after prior induction and consolidation therapies were randomized to receive either OCV-501 (OCV-501 arm, N=68) or placebo (placebo arm, N=65). The median age of the patients was 67 years (range, 60 - 85) and most of them had good performance status (ECOG PS score 0 - 1). The WT1 mRNA level at Day 1 was <50 copies/μg RNA in 37 patients in the OCV-501 arm and 28 patients in the placebo arm. In the evaluation of efficacy, there was no significant difference in DFS between both arms, and the median DFS was 12.1 months and 8.4 months in the OCV-501 arm and placebo arm, respectively. The median OS was 38.5 months and 31 months in the OCV-501 arm and placebo arm, respectively. The results of patient QOL and PS were similar between both arms. Overall, there were no significant safety findings throughout the trial. A majority of the patients in both arms had at least 1 treatment-emergent AE (TEAE); however, most of the TEAEs were mild in severity (Grade 1 or 2). The most frequently reported TEAE was site injection induration.Conclusion: There was no significant difference in DFS between OCV-501- and placebo-treated patients. OCV-501 was found to be generally safe and well-tolerated in elderly AML patients. DisclosuresYamaguchi:Chugai Pharmaceutical Co., Ltd.: Honoraria; Janssen Pharmaceutical KK: Honoraria; Celgene KK: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria. Kiguchi:Teijin Co., Ltd.: Other: Grant; Sanofi K.K., Ltd.: Other: Grant; Celltrion, Inc.: Other: Grant; Taiho Pharmaceutical Co., Ltd.: Other: Grant; SymBio Pharmaceuticals, Ltd.: Other: Grant; Takeda Pharmaceutical Co., Ltd.: Other: Personal fees; Ono Pharmaceutical Co., Ltd.: Other: Personal fees; Astellas Pharmaceutical Co., Ltd.: Other: Grant; Celgene Co., Ltd.: Other: Grant and personal fees; Janssen Pharmaceutical Co., Ltd.: Other: Grant and personal fees; Sumitomo Dainippon Pharmaceutical Co., Ltd.: Other: Grant and personal fees; Novartis Pharmaceutical Co., Ltd.: Other: Grant and personal fees; Nippon Shinyaku Co., Ltd.: Other: Grant and personal fees; Mochida Pharmaceutical Co., Ltd.: Other: Personal fees; Eisai Co., Ltd.: Other: Personal fees; MSD Co., Ltd.: Other: Grant and personal fees; Daiichi Sankyo Pharmaceutical Co., Ltd.: Other: Grant; Chugai Pharmaceutical Co., Ltd: Other: Grant; Bristol Myers Squibb Co., Ltd.: Other: Grant and personal fees; Pfizer Co., Ltd.: Other: Personal fees; Otsuka Pharmaceutical Co., Ltd.: Other: grant and personal fees; Kyowa Hakko Kirin Co., Ltd.: Other: Grant and personal fees. Miyawaki:Novartis Pharma KK: Consultancy; Otsuka Pharmaceutical Co., Ltd.: Consultancy; Astellas Pharma Inc.: Consultancy. Heike:Sumitomo Dainippon Pharma Co., Ltd.: Other: Advisor; Otsuka Pharmaceutical Co., Ltd.: Other: Advisor. Mitsuki:Otsuka Pharmaceutical Co., Ltd.: Employment. Yoshida:Otsuka Pharmaceutical Co., Ltd.: Employment. Liew:Otsuka Pharmaceutical Co., Ltd.: Employment. Naoe:Astellas Pharma Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Toyama Chemical Co., Ltd.: Research Funding.

Immune Monitoring of Vaccine Quality and Persistence of Specific T Cell Responses in Five AML Patients Receiving Extended Dendritic Cell Vaccination Under Compassionate Use

High rates of early disease relapse are observed in patients with acute myeloid leukemia (AML). Due to poor health status, many patients cannot tolerate intensive chemotherapy and/or stem cell transplantation, resulting in a high unmet medical need for new therapy options. Clinical benefit of SCT is associated with immune responses that can control residual leukemia. To replace SCT in non-eligible patients, we designed an autologous dendritic cell (DC) vaccine approach that is given 4 times weekly at the beginning (immunization phase) followed by booster vaccines at week six and then monthly, with the intention to induce immune responses that delay or prevent relapse. The DCs secrete bioactive IL-12, but not IL-10, allowing activation of innate and adaptive responses. T cell responses are directed to two target antigens expressed in AML: Wilm's tumor-1 (WT-1) and preferentially expressed in melanoma (PRAME). Prior to initiation of a Phase I/II study implementing this vaccine approach (EudraCT No.: 2014-003520-44; clinicaltrials.gov No.: NCT02405338), five patients were treated under compassionate use (CU) using DC vaccines prepared according to an approved GMP manufacturing protocol.Here we report immune monitoring studies, exploring quality of vaccines and specificity and persistence of T cell responses. DC vaccines produced in batch lots were cryopreserved in multiple aliquots for thaw prior to application. Extensive characterization showed DCs to be of mature phenotype, with high levels of positive co-stimulatory molecules and fewer negative regulatory molecules. Antigen-loading by electroporation of in vitro transcribed RNA, led to full length intracellular protein expression in most cells. Chemokine-directed migration was measured for all DC preparations. A novel dual-color ELISpot assay showed that DCs of each patient secreted IL-12 but not IL-10 after CD40 ligand stimulation. Despite extensive chemotherapy and impaired hematopoiesis, high quality DC vaccines with essential functions were easily generated from monocytes of these patients.T cell responses to target antigens were assessed with two assays. Intracellular interferon-gamma production was measured in PBL acquired at various time points during treatment. Standard ELISpot assays independently confirmed T cell responses to both antigens. In addition, responses to non-immunizing antigens (hTERT and survivin) were detected in some cases, demonstrating antigen-spreading during treatment. T cell responses to one or both targets were found in 4/5 patients; thus, both self-antigens were immunogenic when presented by polarized mDCs. Persistent vaccination allowed maintenance of T cell responses over extended periods of time in 4/5 patients. Interestingly, antigen-specific responses to over-lapping peptides were only found in two patients when anti-PD-1 blocking antibody was present in the stimulation cultures, thereby enabling T cells to produce cytokine after antigen-specific stimulation.To date 3/5 patients are alive without current signs of relapse. Two patients received DC vaccines for 22 and 30 months and have been followed for 53 and 41 months respectively, since completion of chemotherapy. The third patient had signs of relapse and vaccination was halted after 10 months. This individual was then given allogeneic SCT and remains in CR after 35 months. The two remaining patients are deceased. One patient who showed no T cell activity relapsed after 12 months of vaccination and died at month 20 during chemotherapy. The fifth patient was vaccinated for 24 months without signs of relapse but died due to unrelated heart disease. The ongoing clinical study using extended DC vaccination will provide more information whether persistent vaccination against these self-antigens contributes to immune responses that prevent disease relapse in AML patients. DisclosuresEckl:Medigene Immunotherapies GmbH: Employment. Geiger:Medigene Immunotherapies GmbH: Employment. Schendel:Medigene Immunotherapies GmbH: Employment, Patents & Royalties: DC Vaccines.

Wilm’s tumor-1 (WT1) rs16754 polymorphism and clinical outcome in acute myeloid leukemia

Wilm’s tumor-1 (WT1) rs16754 polymorphism and clinical outcome in acute myeloid leukemia

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

Knock-in of the Wt1 R394W mutation causes MDS and cooperates with Flt3/ITD to drive aggressive myeloid neoplasms in mice.

Wilms tumor 1 (WT1) is a zinc finger transcriptional regulator, and has been implicated as both a tumor suppressor and oncogene in various malignancies. Mutations in the DNA-binding domain of the WT1 gene are described in 10–15% of normal-karyotype AML (NK-AML) in pediatric and adult patients. Similar WT1 mutations have been reported in adult patients with myelodysplastic syndrome (MDS). WT1 mutations have been independently associated with treatment failure and poor prognosis in NK-AML. Internal tandem duplication (ITD) mutations of FMS-like tyrosine kinase 3 (FLT3) commonly co-occur with WT1-mutant AML, suggesting a cooperative role in leukemogenesis. The functional role of WT1 mutations in hematologic malignancies appears to be complex and is not yet fully elucidated. Here, we describe the hematologic phenotype of a knock-in mouse model of a Wt1 mutation (R394W), described in cases of human leukemia. We show that Wt1+/R394W mice develop MDS which becomes 100% penetrant in a transplant model, exhibit an aberrant expansion of myeloid progenitor cells, and demonstrate enhanced self-renewal of hematopoietic progenitor cells in vitro. We crossbred Wt1+/R394W mice with knock-in Flt3+/ITD mice, and show that mice with both mutations (Flt3+/ITD/Wt1+/R394W) develop a transplantable MDS/MPN, with more aggressive features compared to either single mutant mouse model.

Long-term survival of pancreatic cancer patients treated with multimodal therapy combined with WT1-targeted dendritic cell vaccines

ABSTRACTBackground/Aim: Pancreatic ductal adenocarcinoma (PDA) remains one of the most aggressive tumors with a dismally poor prognosis. Although surgical resection remains the only potentially curative treatment, most PDAs are not surgically resectable at diagnosis. Therefore, multimodal therapy is urgently needed to improve the long-term survival of PDA patients.Methods: Six eligible PDA patients underwent multimodal therapy comprising dendritic cells (DCs) pulsed with Wilms’ tumor 1 (WT1) peptide (DC/WT1-I) restricted by the human leukocyte antigen (HLA) class I (A*24:02 or A*02:06) allele, chemotherapy, radiation, and/or surgery. Patient laboratory data, DC/WT1-I-specific delayed-type hypersensitivity (DTH) reactions, and WT1-specific immune responses were analyzed to assess the prognostic markers of multimodal therapy.Results: Compared to 2-treatment type combinations, multimodal therapy involving 3 to 4 treatment types was significantly associated with longer overall survival (p = 0.0177). Moreover, after 7 DC/WT1-I vaccinations, the progression-free survival (PFS) of PDA patients with a neutrophil to lymphocyte ratio (NLR) or C-reactive protein (CRP) level less than the median was superior to that of PDA patients with values above the median (p = 0.0246). PDA patients with an overall survival (OS)>1000 days had significantly more lymphocytes after one DC/WT1-I vaccination course than did those with an OS<1000 days.Conclusion: Multimodal therapy involving the DC/WT1-I vaccination may benefit patients with advanced PDA. However, comparing the limited number of PDA patients in terms of survival is difficult because the patients were at different disease stages and received different treatments. Further studies are needed to evaluate the clinical benefits of this multimodal therapy.

Genetic and epigenetic evolution as a contributor to WT1-mutant leukemogenesis

AbstractGenetic studies have identified recurrent somatic mutations in acute myeloid leukemia (AML) patients, including in the Wilms' tumor 1 (WT1) gene. The molecular mechanisms by which WT1 mutations contribute to leukemogenesis have not yet been fully elucidated. We investigated the role of Wt1 gene dosage in steady-state and pathologic hematopoiesis. Wt1 heterozygous loss enhanced stem cell self-renewal in an age-dependent manner, which increased stem cell function over time and resulted in age-dependent leukemic transformation. Wt1-haploinsufficient leukemias were characterized by progressive genetic and epigenetic alterations, including those in known leukemia-associated alleles, demonstrating a requirement for additional events to promote hematopoietic transformation. Consistent with this observation, we found that Wt1 depletion cooperates with Flt3-ITD mutation to induce fully penetrant AML. Our studies provide insight into mechanisms of Wt1-loss leukemogenesis and into the evolutionary events required to induce transformation of Wt1-haploinsufficient stem/progenitor cells.

Antitumor activity of CAR-T cells targeting the intracellular oncoprotein WT1 can be enhanced by vaccination

AbstractThe recent success of chimeric antigen receptor (CAR)-T cell therapy for treatment of hematologic malignancies supports further development of treatments for both liquid and solid tumors. However, expansion of CAR-T cell therapy is limited by the availability of surface antigens specific for the tumor while sparing normal cells. There is a rich diversity of tumor antigens from intracellularly expressed proteins that current and conventional CAR-T cells are unable to target. Furthermore, adoptively transferred T cells often suffer from exhaustion and insufficient expansion, in part, because of the immunosuppressive mechanisms operating in tumor-bearing hosts. Therefore, it is necessary to develop means to further activate and expand those CAR-T cells in vivo. The Wilms tumor 1 (WT1) is an intracellular oncogenic transcription factor that is an attractive target for cancer immunotherapy because of its overexpression in a wide range of leukemias and solid tumors, and a low level of expression in normal adult tissues. In the present study, we developed CAR-T cells consisting of a single chain variable fragment (scFv) specific to the WT1235-243/HLA-A*2402 complex. The therapeutic efficacy of our CAR-T cells was demonstrated in a xenograft model, which was further enhanced by vaccination with dendritic cells (DCs) loaded with the corresponding antigen. This enhanced efficacy was mediated, at least partly, by the expansion and activation of CAR-T cells. CAR-T cells shown in the present study not only demonstrate the potential to expand the range of targets available to CAR-T cells, but also provide a proof of concept that efficacy of CAR-T cells targeting peptide/major histocompatibility complex can be boosted by vaccination.

Prospective Evaluation for Mutational Frequency of Wilm’s Tumor-1 (WT1) Gene in De Novo Cases of Acute Myeloid Leukaemia

Prospective Evaluation for Mutational Frequency of Wilm’s Tumor-1 (WT1) Gene in De Novo Cases of Acute Myeloid Leukaemia

Podocyte-Specific Induction of Krüppel-Like Factor 15 Restores Differentiation Markers and Attenuates Kidney Injury in Proteinuric Kidney Disease.

Podocyte injury is the hallmark of proteinuric kidney diseases, such as FSGS and minimal change disease, and destabilization of the podocyte's actin cytoskeleton contributes to podocyte dysfunction in many of these conditions. Although agents, such as glucocorticoids and cyclosporin, stabilize the actin cytoskeleton, systemic toxicity hinders chronic use. We previously showed that loss of the kidney-enriched zinc finger transcription factor Krüppel-like factor 15 (KLF15) increases susceptibility to proteinuric kidney disease and attenuates the salutary effects of retinoic acid and glucocorticoids in the podocyte. We induced podocyte-specific KLF15 in two proteinuric murine models, HIV-1 transgenic (Tg26) mice and adriamycin (ADR)-induced nephropathy, and used RNA sequencing of isolated glomeruli and subsequent enrichment analysis to investigate pathways mediated by podocyte-specific KLF15 in Tg26 mice. We also explored in cultured human podocytes the potential mediating role of Wilms Tumor 1 (WT1), a transcription factor critical for podocyte differentiation. In Tg26 mice, inducing podocyte-specific KLF15 attenuated podocyte injury, glomerulosclerosis, tubulointerstitial fibrosis, and inflammation, while improving renal function and overall survival; it also attenuated podocyte injury in ADR-treated mice. Enrichment analysis of RNA sequencing from the Tg26 mouse model shows that KLF15 induction activates pathways involved in stabilization of actin cytoskeleton, focal adhesion, and podocyte differentiation. Transcription factor enrichment analysis, with further experimental validation, suggests that KLF15 activity is in part mediated by WT1. Inducing podocyte-specific KLF15 attenuates kidney injury by directly and indirectly upregulating genes critical for podocyte differentiation, suggesting that KLF15 induction might be a potential strategy for treating proteinuric kidney disease.

Wilms' tumor 1 drives fibroproliferation and myofibroblast transformation in severe fibrotic lung disease.

Wilms' tumor 1 (WT1) is a critical transcriptional regulator of mesothelial cells during lung development but is downregulated in postnatal stages and adult lungs. We recently showed that WT1 is upregulated in both mesothelial cells and mesenchymal cells in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal fibrotic lung disease. Although WT1-positive cell accumulation leading to severe fibrotic lung disease has been studied, the role of WT1 in fibroblast activation and pulmonary fibrosis remains elusive. Here, we show that WT1 functions as a positive regulator of fibroblast activation, including fibroproliferation, myofibroblast transformation, and extracellular matrix (ECM) production. Chromatin immunoprecipitation experiments indicate that WT1 binds directly to the promoter DNA sequence of α-smooth muscle actin (αSMA) to induce myofibroblast transformation. In support, the genetic lineage tracing identifies WT1 as a key driver of mesothelial-to-myofibroblast and fibroblast-to-myofibroblast transformation. Importantly, the partial loss of WT1 was sufficient to attenuate myofibroblast accumulation and pulmonary fibrosis in vivo. Further, our coculture studies show that WT1 upregulation leads to non-cell autonomous effects on neighboring cells. Thus, our data uncovered a pathogenic role of WT1 in IPF by promoting fibroblast activation in the peripheral areas of the lung and as a target for therapeutic intervention.

Overexpression of Wilms' tumor 1 in skin lesions of psoriasis is associated with abnormal proliferation and apoptosis of keratinocytes.

Psoriasis vulgaris (PV) is a chronic inflammatory skin disease, which is characterized by the abnormal proliferation and apoptosis of keratinocytes. Previous studies have demonstrated that transcription factor Wilms' tumor 1 (WT1) is involved in a number of pathophysiological processes, including organ development, tumorigenesis and cell proliferation. However, the role of WT1 in PV remains unclear. In the present study, WT1 expression was analyzed by reverse transcription‑quantitative polymerase chain reaction and western blot analyses. WT1 was stably overexpressed or inhibited in HaCaT cells using Lipofectamine® 2000, and cell proliferation and apoptosis were determined using a Cell Counting Kit‑8 or Fluorescein Isothiocyanate Annexin V Apoptosis Detection kit II, respectively. We demonstrated that compared with normal controls, the mRNA and protein expression levels of WT1 were significantly increased in non‑lesional skins (human, P<0.0001 and P=0.0291, respectively; mouse, P=0.0020 and P=0.0150, respectively) and lesional skins (human, P<0.0001 and P=0.0060, respectively; mouse, P=0.0010 and P=0.0172, respectively) of patients with PV, in addition to the imiquimod (IMQ)‑induced psoriasis‑like mouse model. WT1 mRNA and protein expression levels in lesional skins were slightly increased compared with those in non‑lesional skins from patients with psoriasis (P=0.2510 and P=0.1690, respectively) and IMQ‑treated mice (P=0.9590 and P=0.2552, respectively), although there were no statistical differences. Knockdown of WT1 inhibited the proliferation of HaCaT cells [day (D)4, P=0.0454; D5, P=0.0021] and promoted their apoptosis (P=0.0007), while overexpressing WT1 exhibited the opposite effects (proliferation D3, P=0.0216; D4, P=0.0356; D5, P=0.0188; apoptosis, P=0.0003). Furthermore, it was identified that the inflammatory cytokines interleukin‑17A (IL‑17A), interferon‑γ and IL‑22 induced the overexpression of WT1 in HaCaT cells. The results of the present study suggested that inflammatory cytokine‑induced WT1 overexpression may promote the formation of psoriatic skin lesions via regulation of the proliferation and apoptosis of keratinocytes.

Ancillary studies in pleural, pericardial, and peritoneal effusion cytology.

Pleural, pericardial, and peritoneal effusion specimens present diagnostic challenges and clinical opportunities for cytology. For the patient, these specimens may be the first diagnosis of malignancy or the first sign of disease recurrence. This review aims to provide useful approaches with which to resolve key difficulties in cytologic diagnosis through the use of ancillary studies, focusing on immunohistochemistry. These approaches are suggested in concert with clinical, radiographic, and morphologic findings. The differentiation of reactive mesothelial cells from malignant mesothelioma and adenocarcinoma is a recurring theme, and Wilms tumor 1 (WT1)/AE1/AE3, claudin 4, and BRCA1-associated protein 1 (BAP1) immunostains are useful new tools in the armamentarium. A targeted workup is suggested for patients with no known primary site or with multiple prior malignancies. Molecular and other biomarker testing can be performed on even modestly cellular fluid specimens and may allow patients to benefit from targeted therapy without the need for additional tissue biopsies. Cancer Cytopathol 2018;000:000-000. © 2018 American Cancer Society.

METTL3 regulates WTAP protein homeostasis

The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumors, including, acute myeloid leukemia (AML), where it plays an oncogenic role by interacting with different proteins involved in RNA processing and cell proliferation. In addition, WTAP is also a regulator of the nuclear complex required for the deposition of N6-methyladenosine (m6A) into mRNAs, containing the METTL3 methyltransferase. However, it is not clear if WTAP may have m6A-independent regulatory functions that might contribute to its oncogenic role. Here, we show that both knockdown and overexpression of METTL3 protein results in WTAP protein upregulation, indicating that METTL3 levels are critical for WTAP protein homeostasis. However, we show that WTAP upregulation is not sufficient to promote cell proliferation in the absence of a functional METTL3. Therein, these data indicate that the reported oncogenic function of WTAP is strictly connected to a functional m6A methylation complex.