Wilms Tumor 1 Research Articles

Abstract P124: Post Acute Infection Of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Leads To Systemic Inflammation And Podocyte Injury.

Introduction: COVID-19 attributed to SARS-CoV2 infection is a world-wide pandemic. SARS-CoV2 has been associated with cardiovascular disease and diabetes. At a cellular level, the infection causes endothelial cell dysfunction (ECD), which is manifested by entities such as microvascular thrombosis. ECD is quite common in type 2 diabetes mellitus (T2DM) where the renal podocyte dysfunction is often an early manifestation of microvascular complication. In this study we explored whether presence of hyperglycemia predisposes to increased SARS-CoV2 infection and whether co-morbid presence of COVID-19 and hyperglycemia predisposes to cardio-metabolic complications such as diabetic kidney disease (DKD). To estimate kidney damage we evaluated albuminuria and podocyte markers in urine exosomes from SARS-CoV2 patients at 10 days, 6 months and 12 months post COVID-19 infection. Methods: Blood and Urine samples from SARS-CoV2 patients post acute phase of infection were procured from the core facility. Peripheral blood mononuclear cells (PBMNCs) and urine exosomes were isolated and podocyte protein markers such as Nephrin (Nep), Podocalyxin (PODXL) and Wilm’s Tumour-1 (WT-1) were identified by western blot analysis. Results: Our results showed that human kidney cells on exposure to hyperglycemia (5 vs 25mM) upregulates expression of TMPRSS2 gene, a key receptor for SARS-CoV2, by 5 fold. Next we examined blood and urine samples from COVID-19, subjects (n=16). As all examined subjects had blood glucose levels above 400mg%, we used samples from T2DM subjects with no COVID-19 infection, as a comparator. COVID-MNCs showed persistent over-expression of IL-6 and TNFa (4-fold) even at 12 months post infection. Urine-exosomal-Nephrin protein band expression was 10-fold higher than T2DM-No-COVID urine-exosomal-Nephrin protein band Similar trend was noted on estimating PODXL and WT-1. Conclusions: A persistent inflammatory marker over-expression at 12 months in COVID samples indicate possible long standing renal damage. Increased podocyte specific protein loss at 12 months compared to samples from T2DM subjects with similar GFR also indicates persistent kidney damage which may lead to renal failure and hypertension.

The essential glucose transporter GLUT1 is epigenetically upregulated by C/EBPβ and WT1 during decidualization of the endometrium

Human endometrial stromal cells (ESCs) differentiate into decidual cells by the action of progesterone, which is essential for implantation and maintenance of pregnancy. We previously reported that glucose uptake by human ESCs increases during decidualization and that glucose is indispensable for decidualization. Although glucose transporter 1 (GLUT1) is upregulated during decidualization, it remains unclear whether it is involved in glucose uptake. Here, we attempted to determine the role of GLUT1 during decidualization as well as the factors underlying its upregulation. ESCs were incubated with cAMP to induce decidualization. Knockdown of GLUT1 suppressed cAMP-increased glucose uptake and the expressions of specific markers of decidualization, IGF-binding protein-1 (IGFBP-1), and prolactin (PRL). To investigate the regulation of GLUT1 expression, we focused on CCAAT enhancer-binding protein β (C/EBPβ) and Wilms’ tumor 1 (WT1) as the upstream transcription factors regulating GLUT1 expression. Knockdown of either C/EBPβ or WT1 suppressed cAMP-increased GLUT1 expression and glucose uptake. cAMP treatment also increased the recruitment of C/EBPβ and WT1 to the GLUT1 promoter region. Interestingly, cAMP increased the H3K27 acetylation (H3K27ac) and p300 recruitment in the GLUT1 promoter region. Knockdown of C/EBPβ or WT1 inhibited these events, indicating that both C/EBPβ and WT1 contribute to the increase of H3K27ac by recruiting p300 to the GLUT1 promoter region during decidualization. These findings indicate that GLUT1 is involved in glucose uptake in ESCs during decidualization, thus facilitating the establishment of pregnancy.

AML-237: Wilms Tumor 1 (WT-1) Gene Methylation as an Epigenetic Biomarker in Acute Myeloid Leukemia

Context: Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy characterized by different genetic and epigenetic aberrations. Several epigenetics modifications were observed in the promoter regions of the cellular epigenome, including hypomethylation or hypermethylation of CpG islands during the onset and progression of AML. DNA methylation is an epigenetic modification that regulates gene expression and plays a pivotal role in tumorigenesis, including AML. Wilms tumor 1 (WT-1) is a transcription factor involved in the differentiation and proliferation of AML. The clinical relevance of WT-1 gene expression and its underlying epigenetic alterations has not been fully addressed in AML. Objective: This work aimed to investigate promoter methylation and mRNA expression of WT-1 gene in AML. Patients: Bone marrow samples were collected at the time of diagnosis and after completion of induction chemotherapy from 10 newly diagnosed AML patients. Five non-malignant BM samples were recruited as controls. Methods: Quantitative assessment of WT-1 gene transcripts was performed using real-time PCR. In addition, methylation-specific PCR was done to evaluate the promoter methylation status of the WT-1 gene in AML. Results: Robust promoter hypermethylation and higher expression of the WT-1 gene were observed in AML patients at time of diagnosis. In contrast, hypomethylation and lower WT-1 gene expression were observed after completion of induction therapy (p<0.001). Conclusions: This is the first report, to our knowledge, regarding WT-1 gene overexpression/hypermethylation signature as a characteristic marker that correlates with leukemic burden in AML among the Indian population. Increased expression and methylation signature of the WT-1 gene can be used as a promising molecular marker for diagnosis, clinical progression of the diseases, or monitoring response to treatment, as well as a target for the development of novel therapeutic approaches. Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy characterized by different genetic and epigenetic aberrations. Several epigenetics modifications were observed in the promoter regions of the cellular epigenome, including hypomethylation or hypermethylation of CpG islands during the onset and progression of AML. DNA methylation is an epigenetic modification that regulates gene expression and plays a pivotal role in tumorigenesis, including AML. Wilms tumor 1 (WT-1) is a transcription factor involved in the differentiation and proliferation of AML. The clinical relevance of WT-1 gene expression and its underlying epigenetic alterations has not been fully addressed in AML. This work aimed to investigate promoter methylation and mRNA expression of WT-1 gene in AML. Bone marrow samples were collected at the time of diagnosis and after completion of induction chemotherapy from 10 newly diagnosed AML patients. Five non-malignant BM samples were recruited as controls. Quantitative assessment of WT-1 gene transcripts was performed using real-time PCR. In addition, methylation-specific PCR was done to evaluate the promoter methylation status of the WT-1 gene in AML. Robust promoter hypermethylation and higher expression of the WT-1 gene were observed in AML patients at time of diagnosis. In contrast, hypomethylation and lower WT-1 gene expression were observed after completion of induction therapy (p<0.001). This is the first report, to our knowledge, regarding WT-1 gene overexpression/hypermethylation signature as a characteristic marker that correlates with leukemic burden in AML among the Indian population. Increased expression and methylation signature of the WT-1 gene can be used as a promising molecular marker for diagnosis, clinical progression of the diseases, or monitoring response to treatment, as well as a target for the development of novel therapeutic approaches.

Poster: AML-237: Wilms Tumor 1 (WT-1) Gene Methylation as an Epigenetic Biomarker in Acute Myeloid Leukemia

Poster: AML-237: Wilms Tumor 1 (WT-1) Gene Methylation as an Epigenetic Biomarker in Acute Myeloid Leukemia

Proteolysis-Targeting Chimeras Enhance T Cell Bispecific Antibody-Driven T Cell Activation and Effector Function through Increased MHC Class I Antigen Presentation in Cancer Cells.

The availability of Ags on the surface of tumor cells is crucial for the efficacy of cancer immunotherapeutic approaches using large molecules, such as T cell bispecific Abs (TCBs). Tumor Ags are processed through intracellular proteasomal protein degradation and are displayed as peptides on MHC class I (MHC I). Ag recognition through TCRs on the surface of CD8+ T cells can elicit a tumor-selective immune response. In this article, we show that proteolysis-targeting chimeras (PROTACs) that target bromo- and extraterminal domain proteins increase the abundance of the corresponding target-derived peptide Ags on MHC I in both liquid and solid tumor-derived human cell lines. This increase depends on the engagement of the E3 ligase to bromo- and extraterminal domain protein. Similarly, targeting of a doxycycline-inducible Wilms tumor 1 (WT1)-FKBP12F36V fusion protein, by a mutant-selective FKBP12F36V degrader, increases the presentation of WT1 Ags in human breast cancer cells. T cell-mediated response directed against cancer cells was tested on treatment with a TCR-like TCB, which was able to bridge human T cells to a WT1 peptide displayed on MHC I. FKBP12F36V degrader treatment increased the expression of early and late activation markers (CD69, CD25) in T cells; the secretion of granzyme β, IFN-γ, and TNF-α; and cancer cell killing in a tumor-T cell coculture model. This study supports harnessing targeted protein degradation in tumor cells, for modulation of T cell effector function, by investigating for the first time, to our knowledge, the potential of combining a degrader and a TCB in a cancer immunotherapy setting.

Cholesterol is required for transcriptional repression by BASP1

Lipids are present within the cell nucleus, where they engage with factors involved in gene regulation. Cholesterol associates with chromatin in vivo and stimulates nucleosome packing in vitro, but its effects on specific transcriptional responses are not clear. Here, we show that the lipidated Wilms tumor 1 (WT1) transcriptional corepressor, brain acid soluble protein 1 (BASP1), interacts with cholesterol in the cell nucleus through a conserved cholesterol interaction motif. We demonstrate that BASP1 directly recruits cholesterol to the promoter region of WT1 target genes. Mutation of BASP1 to ablate its interaction with cholesterol or the treatment of cells with drugs that block cholesterol biosynthesis inhibits the transcriptional repressor function of BASP1. We find that the BASP1-cholesterol interaction is required for BASP1-dependent chromatin remodeling and the direction of transcription programs that control cell differentiation. Our study uncovers a mechanism for gene-specific targeting of cholesterol where it is required to mediate transcriptional repression.

The prognostic impact of Wilms tumor-1 polymorphism (rs16754) and human myeloid inhibitory C-type lectin-like receptor expression in cytogenetically normal-acute myeloid leukemia

BackgroundThere are several genetic mutations that carry prognostic and predictive values in acute myeloid leukemia (AML). They are also implicated in disease pathogenesis and patient outcome. They can be a target of novel therapies for AML. The aim of the current study was to investigate prognostic value of Wilms’ tumor-1 (WT1) genotypes and human myeloid inhibitory C-type lectin-like (hMICL) receptor expression in normal-cytogenetic group of patients with AML. Genotyping of WT1 mutations was done by Rotor Gene real-time polymerase chain reaction (PCR) while hMICL expression was detected using phycoerythrin (PE)-conjugated mouse monoclonal anti-human (MoAbs) by flow cytometry.ResultsSixty-three patients with cytogenetically normal AML (CN-AML) were included in the study. The alternate allele of WT1 single nucleotide polymorphism (SNP) rs16754 was found in 26.89%. At day 28 of therapy, complete remission was achieved in 100% of cases harboring mutant AG plus GG genotypes but only in 6.38% of cases harboring wild genotype (AA). After 6 months, 88.23% of patients harboring WT1 mutant genotype maintained complete remission, while only 23.40% of patients with wild type showed complete remission. The overall survival in patients harboring mutant WT1 genotypes was significantly longer than in those who carried the wild type gene (P-value, 0.001). Additionally, hMICL was overexpressed in approximately 87.3% of AML cases and inversely related to complete response. Similarly, overall survival was significantly shorter in patients with positive hMICL (P-value, 0.001).ConclusionMutant WT1 genotypes (SNP rs16754) were conversely, associated with complete response, and hMICL overexpression had poor prognostic value in AML.

Clinicopathological significance of WT1 expression in invasive breast carcinoma with >90% mucinous component

AimsThis study was aimed to investigate the clinicopathological significance of immunohistochemical (IHC) Wilm’s tumour 1 (WT1) expression in invasive breast carcinoma with >90% mucinous components.MethodsOne hundred specimens of invasive breast...

Expression of the Tumor Suppressor WT1 Is Induced By PML-Rara in Acute Promyelocytic Leukemia. Â

Inactivating mutations in Wilms Tumor 1 (WT1) are found in 5-10% of cases of acute myeloid leukemia (AML). Paradoxically, wild type WT1 is expressed highly in most cases of AML compared to normal hematopoietic progenitors, and high levels of WT1 have been associated with poor outcomes. Therefore, it is not yet clear whether loss-of-function WT1 mutations-or paradoxically, wild type WT1 overexpression-both promote disease progression in AML. We performed a genome-wide screen for mutations in acute promyelocytic leukemia (APL), a morphologic subtype of AML characterized by the PML-RARA fusion. We identified WT1 mutations in 11/42 patients, suggesting that WT1 mutations may cooperate specifically with PML-RARA in APL. Further, expression analysis demonstrated a high level of wild type WT1 expression in non-mutated cases. To test the effect of Wt1 inactivation in a mouse model of APL, we bred mice carrying a floxed Wt1 allele (give Baylor reference) to transgenic mice expressing tamoxifen-inducible Cre recombinase (ERT2-Cre). Tamoxifen treatment leads to deletion of the C-terminal zinc finger domains of Wt1, mimicking some of the mutations that are commonly found in AML/APL cases. These mice were crossed with mice expressing the PML-RARA fusion cDNA in early myeloid progenitors with Cathepsin G (Ctsg) regulatory sequences (Ctsg-PML-RARA). Bone marrow cells from Ctsg-PML-RARA x Wt1fl/fl x ERT2-Cre mice were transplanted into irradiated recipients. Tamoxifen-treated mice (i.e. Wt1 deficient) developed APL with a significantly shortened latency compared to untreated controls (198 days vs. endpoint not reached, N=8-10 each group, p=.0001), suggesting that Wt1 loss-of-function mutations can cooperate with PML-RARA to drive APL. Next, to test the effect of wild type Wt1 overexpression on hematopoietic cells, bone marrow cells from wild type or Ctsg-PML-RARA mice was transduced with retroviruses expressing full length Wt1 cDNA or an empty vector control, and subjected to serial replating. As previously reported, untransduced bone marrow cells from Ctsg-PML-RARA mice were able to form colonies in methylcellulose week after week, while cells overexpressing wild type Wt1 were selected against during serial replating, as demonstrated by a gradual reduction in GFP percentage and Wt1 expression, as measured by western blotting. Interestingly, in contrast to human AML/APL, Wt1 expression could not be detected in a panel of murine tumors with initiating mutations in DNMT3A, PML-RARA, or MLL-AF9 . Taken together, these findings suggest that Wt1 expression suppresses APL development in mice.To reconcile these findings with the high level of WT1 expression in human AML/APL, we speculated that in human progenitor cells, WT1 expression may be induced by leukemia-associated oncogenes (like PML-RARA) as a physiologic, inhibitory response to oncogenic stress. In this case, subsequent inactivating mutations in WT1 would provide an additional growth advantage. Given the observed interspecies difference between WT1/Wt1 expression levels in mouse and human leukemia cells, we used human umbilical cord blood-derived CD34+ cells transduced with retroviruses expressing PML-RARA to model early events in human APL development. CD34+ cells expressing PML-RARA share some characteristics of fully transformed APL cells, including enhanced proliferation in liquid culture, and reduced expression of the myeloid maturation markers CD11b and CD14. WT1 mRNA expression is low in freshly isolated CD34+ cells, but rapidly increases in cells expressing PML-RARA over two weeks in culture (10.5-fold increase compared to empty-vector transduced controls, N=3 separate experiments, p<0.05). Western blotting confirmed robust induction of WT1 protein in transformed cells compared to empty-vector transduced controls, suggesting that WT1 expression is a normal consequence of PML-RARA activation in human hematopoietic progenitor cells. In summary, these findings support a cooperating role for WT1 mutations in APL development, and a potential mechanism to explain the high level of wild type WT1 expression observed in APL cells. Further experiments will be required to determine the downstream mechanisms by which WT1 antagonizes APL development, and whether these observations are generalizable to other AML subtypes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of Cross-Reactive Targets of TCR-Based Therapeutic Agents within the Human Proteome

TCR-based therapeutic cells and agents (antigen-adapted T cell infusions, TCR-engineered T cells, ImmTACs, and TCR mimic antibodies) have emerged as a new class of effective cancer therapeutics. Cross-reactivities of these agents to off-target cells and tissues are poorly understood, difficult to predict, and have resulted in serious, sometimes fatal adverse reactions. There are no reliable techniques for determining the on-target and off-target reactivity of these agents. We sought to develop a method for predicting reactivities of TCR-based agents, starting with three well-defined tools and systems. Wilms Tumor 1 (WT1) and Preferentially expressed antigen in melanoma (PRAME) are overexpressed protein-coding genes in many hematologic malignancies with restricted expression in normal tissue. The ability to target cells expressing these genes is limited because their protein products are intracellular and no small molecules to these drugs exist. T cell receptor (TCR) mimic antibodies (TCRm) can bind to 8-11aa peptide antigens in the context of major histocompatibility complexes (MHC) on the surface of cells. The peptide MHC (pMHC) targets of TCR and TCRm can be derived from wild-type or mutated, intracellular or extracellular proteins, thereby allowing TCRm therapeutics to access a universe of proteins that traditional antibodies cannot reach. We have generated two TCRm antibodies, one directed to a HLA-A*02:01 peptide from the Wilms Tumor 1 protein (WT1 126-134: RMFPNAPYL) and one to the Preferentially Expressed Antigen In Melanoma (PRAME 300-309: ALYVDSLFFL) protein. Like the TCR on T cells, TCRm can cross-react with off-target peptides that share sequence homology. In order to identify endogenous cross-reactive peptides for these therapeutic drugs, we have created a minigene-based method (termed “PresentER”) to genetically encode pMHC on model target cells. PresentER is an easily clonable, Illumina-sequencing compatible genetic system capable of expressing a single MHC-I peptide ligand (8-12 amino acid long). This system is highly specific to the encoded linear amino acid sequence and does not require proteasome cleavage, peptide transport or peptide processing for presentation on MHC-I. PresentER expressing cells can stimulate T cells, bind to TCR and TCRm and be used in pooled screens to discover ligands of TCR and TCRm. We have cloned a library of ~12,000 exomic HLA-A*02:01 ligands and performed a pooled screen of pMHC that can be recognized by the TCRm. The utility of PresentER was validated in its first application to find dozens of MHC-I ligands encoded in the human proteome that were cross-reactive with the ESK1 TCRm. The PresentER screen revealed MHC-I ligands of ESK1 and Pr20 that were not predictable by other methods. PresentER may be used to discover the ligandome of T cell receptors and T cell receptor mimic antibodies in a native, cell-based context. DisclosuresGejman:Memorial Sloan Kettering Cancer Center: Patents & Royalties: US 62/62/395,577. Dao:Memorial Sloan Kettering Cancer Center: Patents & Royalties: WO2012135854 A2. Scheinberg:Memorial Sloan Kettering Cancer Center: Patents & Royalties: WO2012135854 A2, US Provisional 62/62/395,577; Actinium Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Eureka Therapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Pulmonary Hypertension from Cardiorenal Sequelae in an 18-Year-Old 46, XY Phenotypically Female Patient with Denys-Drash Syndrome: Case Report and Review of Literature

Denys-Drash Syndrome (DDS) is a rare cause of end-stage renal disease (ESRD) stemming from genetic mutations in the Wilms’ tumor 1 (WT1) gene. Albeit uncommon, it and other congenital nephropathies (e.g., Frasier syndrome) may warrant a clinical workup of cardiovascular disease in symptomatic patients with cardiopulmonary deficits, as the latter is associated with renal dysfunction given the multifarious physiologic roles and nodes of action concerning the kidneys. These include not only serving as a site of neural and hormonal stimuli that affect systemic equilibrium, but the regulation of blood pressure and osmolality as well. The cardiorenal demise classically seen in later stages of chronic kidney disease (CKD) in relatively older adults may arise earlier in these patients. We report the case of an 18-year-old 46, XY phenotypic female with DDS who complained of increasingly nettlesome dyspnea. Initially, she presented with a low index of suspicion for diminished cardiopulmonary fitness. However, further clinical workup which included catheterization and cardiac imaging was significant for findings of pulmonary hypertension.

The Expression of Transcription Factors in Fetal Lamb Kidney.

(1) Background: Renal development involves frequent expression and loss of transcription factors, resulting in the activation of genes. Wilms’ tumor 1 (WT1), hepatocyte nuclear factor-1-beta (HNF1β), and paired box genes 2 and 8 (Pax2 and Pax8) play an important role in renal development. With this in vivo study, we examined the period and location of expression of these factors in renal development. (2) Methods: Fetal lamb kidneys (50 days from gestation to term) and adult ewe kidneys were evaluated by hematoxylin and eosin staining. Serial sections were subjected to immunohistochemistry for WT1, HNF1β, Pax2, and Pax8. (3) Results: Pax2, Pax8, and HNF1β expression was observed in the ureteric bud and collecting duct epithelial cells. We observed expression of WT1 alone in metanephric mesenchymal cells, glomerular epithelial cells, and interstitial cells in the medullary rays and Pax8 and HNF1β expression in tubular epithelial cells. WT1 was highly expressed in cells more proximal to the medulla in renal vesicles and in C- and S-shaped bodies. Pax2 was expressed in the middle and peripheral regions, and HNF1β in cells in the region in the middle of these. (4) Conclusions: WT1 is involved in nephron development. Pax2, Pax8, and HNF1β are involved in nephron maturation and the formation of peripheral collecting ducts from the Wolffian duct.

Novel combined variants of WT1 and TET2 in a refractory and recurrent AML patient

BackgroundSomatic mutations in Wilms' tumor 1 (WT1) and tet methylcytosine dioxygenase 2 (TET2) genes were separately perceived as contributors to hematopoietic disorders and usually thought to have a mutually exclusive effect in acute myeloid leukemia (AML). However, we found novel WT1 and TET2 variants persistently co-existed in a refractory and recurrent AML patient with t(9;11)(p21.3;q23.3); KMT2A-MLLT3, and were only detectable genetic alteration in early recurrence. Hence, these two novel variants were further investigated in patient’s family, and the potential effect on disease progression was evaluated at follow-up.Case presentationA 27-year-old male was diagnosed with AML, having t(9;11)(p21.3;q23.3); KMT2A-MLLT3, accompanied by WT1 (NM_024426.6:exon7:c.1109G>C:p.Arg370Pro) and TET2 (NM_001127208.3:exon11:c.5530G>A:p.Asp1844Asn) variants. After two cycles of induction chemotherapy, complete remission was achieved. A consolidation treatment was then completed. However, the evaluation of the bone marrow revealed that early recurrence, WT1 (p.Arg370Pro) and TET2 (p.Asp1844Asn) variants still detectable, instead of KMT2A-MLLT3. Subsequently, these two variants were proved to be germline variants, which inherited from father and mother respectively. And the patient's elder brother also carried TET2 (p.Asp1844Asn) variant. A sequential allogeneic HLA-matched sible hematopoietic stem cell transplantation (allo-HSCT) was carried out, and the donor is the patient's elder brother, the original two variants of patient were replaced by the donor-derived TET2 (p.Asp1844Asn) variant after allo-HSCT; the patient has remained in complete remission with regular follow-up.ConclusionsIn brief, it is firstly reported that WT1 p.Arg370Pro and TET2 p.Asp1844Asn variants co-existed in a refractory and recurrent AML patient by inheritance. These two variants of the patient were replaced with donor-derived TET2 p.Asp1844Asn after allo-HSCT, and the patient has remained in complete remission with regular follow-up.

Overexpression of Wilms tumor 1 promotes IL-1β expression by upregulating histone acetylation in keratinocytes

Psoriasis is a common inflammatory skin disease. Infiltration of inflammatory cells and excessive proliferation of keratinocytes are the histopathological markers of psoriasis. The transcription factor Wilms Tumor 1 (WT1) is overexpressed in several tumor types, and plays an important part in the proliferation and apoptosis of cells. Studies have found that, compared with normal skin, WT1expression in the skin lesions of patients with psoriasis are increased significantly. Knockdown of WT1 inhibited the proliferation of a human epidermal keratinocyte cell line (HaCaT cells) and promoted their apoptosis, whereas WT1 overexpression exhibited the opposite effect. WT1 was overexpressed or inhibited in HaCaT cells by transfection with the WT1 plasmid or WT1 small interferring RNA (siRNA) using Lipofectamine 2000. Transcriptome sequencing and bioinformatics analysis revealed significant differences in IL-1β expression between the experimental group and control group. Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assays showed that expression of IL-1β and WT1 were consistent. Subsequently, IL-1β was demonstrated to be a target of WT1 by chromatin immunoprecipitation (ChIP)-sequencing and luciferase reporter assay. ChIP-qPCR showed that WT1 regulated IL-1β expression by altering acetylation. Expression of WT1 mRNA was positively correlated with expression of IL-1β mRNA in psoriatic skin lesions. Our study suggested that WT1 likely promotes psoriasis development by regulating its target gene IL-1β, which shows high expression in psoriatic lesions and is involved in psoriasis development. These findings provide a new target for psoriasis treatment.

Prognostic impact of pretransplant measurable residual disease assessed by peripheral blood WT1‐mRNA expression in patients with AML and MDS

As peripheral blood (PB) Wilm's Tumor 1 (WT1)-mRNA expression is established as MRD-marker during conventional AML chemotherapy, impact of pretransplant WT1 expression remains unclear. Therefore, we aimed to assess prognostic impact of pretransplant WT1 expression on post-transplant outcome in patients with AML/MDS. In 64 AML/MDS patients, pretransplant WT1 expression was retrospectively analyzed using a standardized assay offering high sensitivity, specificity, and a validated cut-off. Patients were divided into three groups determined by pretransplant remission and WT1 expression. Post-transplant outcome of these groups was compared regarding cumulative incidence of relapse (CIR), relapse-free (RFS), and overall survival (OS). Pretransplant forty-six patients (72%) showed hematologic remission, including 21 (46%) MRD-negative and 25 (54%) MRD-positive patients indicated by WT1 expression, while 18 refractory patients (28%) showed active disease. Two-year estimates of post-transplant CIR, RFS, and OS were similar in MRD-positive (61%, 37%, 54%) and refractory patients (70%, 26%, 56%), but significantly inferior compared with MRD-negative patients (10%, 89%, 90%). After multivariable adjustment, pretransplant MRD negativity measured by WT1 expression retained its prognostic impact on CIR (P=.008), RFS (P=.005), and OS (P=.049). PB WT1 expression represents a useful method to estimate pretransplant MRD, which is highly predictable for post-transplant outcome and may help improving peri-transplant management in AML/MDS patients.

Non-transplantable cord blood units as a source for adoptive immunotherapy of leukaemia and a paradigm of circular economy in medicine.

Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.

Dendritic cell vaccination combined with a conventional chemotherapy for patients with relapsed or advanced pancreatic ductal adenocarcinoma: a single-center phase I/II trial.

The prognosis of patients with advanced pancreatic cancer is poor. In the present phase I/II study, we have evaluated the safety and the feasibility of Wilms' tumor 1 (WT1) and/or mucin1 (MUC1) peptide-pulsed dendritic cell (DC) vaccination in combination with chemotherapy in patients with advanced or relapsed pancreatic ductal adenocarcinoma (PDAC). Sixty-five eligible patients were enrolled. No severe adverse events related to the vaccinations were observed. Objective response rate and disease control rate was 12.3% and 50.8%, respectively. Median progression-free survival and overall survival were 4.9 and 9.6months, respectively. DC vaccinations augmented WT1- and MUC1-specific immunity which might be related to clinical outcome. These results indicate that DC-based immunotherapy combined with a conventional chemotherapy is safe and feasible for patients in advanced stage of PDAC.

Inhibition of Cytochrome P450 1B1 Prevents Hepatic Stellate Cell Activation and Liver Fibrosis

Background : Liver fibrosis often results from chronic liver injury and it is characterized by the accumulation of extracellular matrix. Activation of the hepatic stellate cell (HSC) is a key fibrogenic event. Cytochrome P450 1b1 (Cyp1b1) is a heme-thiolate monooxygenase involved in the metabolism of endogenous compounds and xenobiotics. The goal of this study is to determine whether and how Cyp1b1 plays a role in hepatic stellate cell (HSC) activation and liver fibrosis. Methods : The effects of Cyp1b1 on HSC activation and fibrogenesis were assessed by real-time PCR, immunofluorescence, and RNA-seq analysis. Wild-type (WT) and Cyp1b1 knockout (Cyp1b1-/-) mice were subjected to thioacetamide (TAA), carbon tetrachloride (CCl4), or bile duct ligation (BDL) models of liver fibrosis, and the fibrosis phenotypes were analyzed at the histology and gene expression levels. Hepatic retinoid levels were measured by liquid chromatography-mass spectrometry (LC-MS/MS). Results : Cyp1b1 is highly expressed in HSCs. The expression of Cyp1b1 was elevated in fibrotic mouse and human livers, as well as in activated mouse and human HSCs. In contrast, down-regulation or pharmacological inhibition of Cyp1b1 by 2,4,3′,5′-tetramethoxystilbene (TMS) attenuated HSC activation in vitro and in vivo. Cyp1b1-/- mice or WT mice treated with TMS were protected from liver fibrosis. Mechanistically, the fibrogenic induction of Cyp1b1 is likely mediated by the transcriptional factor wilms' tumor 1 (WT1). The pro-fibrogenic activity of Cyp1b1 may have been accounted for by its metabolism of retinoids in HSCs. Conclusion : Our study has uncovered an important endobiotic function of Cyp1b1 in promoting liver fibrosis. Cyp1b1 promotes HSC activation and liver fibrogenesis by facilitating retinoids depletion. Pharmacological inhibition of Cyp1b1 might be a potential strategy for the treatment of liver fibrosis.

Podocytes Specific Deletion of PKM2 Ameliorates LPS‐induced Podocyte Injury through Regulation of Beta‐Catenin Activity

Background Acute kidney injury (AKI) is associated with a severe decline in kidney function caused by abnormalities within the podocytes' glomerular matrix. Recently, AKI has been linked to alterations in glycolysis and the activity of glycolytic enzymes, including pyruvate kinase M2 (PKM2). However, the contribution of this enzyme to AKI remains largely unexplored. Methods Cre-loxP technology was used to examine the effects of PKM2 specific deletion in podocytes on the activation status of key signaling pathways involved in the pathophysiology of AKI by lipopolysaccharides (LPS). In addition, we used lentiviral shRNA to generate murine podocytes deficient in PKM2 and investigated the molecular mechanisms mediating PKM2 actions in vitro. Results Specific PKM2 deletion in podocytes ameliorated LPS-induced protein excretion and alleviated LPS-induced alterations in blood urea nitrogen and serum albumin levels. In addition, PKM2 deletion in podocytes alleviated LPS-induced renal injury and protected mice from LPS-induced pro-inflammatory cytokine expression. At the molecular level, PKM2 deficiency in podocytes alleviated LPS-induced inflammation, endoplasmic reticulum stress, and apoptosis, but exacerbated LPS-induced autophagy. In vitro, PKM2 knockdown in murine podocytes suppressed LPS-induced apoptosis. These effects were concomitant with a reduction in LPS-induced activation of β-catenin and the loss of Wilms’ Tumor 1 (WT1) and nephrin. Notably, the overexpression of a constitutively active mutant of β-catenin abolished the protective effect of PKM2 knockdown. Conversely, Knockdown cells reconstituted with the phosphotyrosine binding–deficient PKM2 mutant (K433E) recapitulated the effect of PKM2 depletion on LPS-induced apoptosis, β-catenin activation, and reduction in WT1 expression. Conclusion Our data demonstrate that PKM2 plays a key role in podocytes injury and suggest that targetting PKM2 in podocytes could serve as a promising therapeutic strategy for AKI.

Wilms Tumor 1 Mutations Are Independent Poor Prognostic Factors in Pediatric Acute Myeloid Leukemia.

The prognostic impact of Wilms tumor 1 (WT1) mutations remains controversial for patients with acute myeloid leukemia (AML). Here, we aimed to determine the clinical implication of WT1 mutations in a large cohort of pediatric AML. The clinical data of 870 pediatric patients with AML were downloaded from the therapeutically applicable research to generate effective treatment (TARGET) dataset. We analyzed the prevalence, clinical profile, and prognosis of AML patients with WT1 mutations in this cohort. Our results showed that 6.7% of total patients harbored WT1 mutations. These WT1 mutations were closely associated with normal cytogenetics (P<0.001), FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD) mutations (P<0.001), and low complete remission induction rates (P<0.01). Compared to the patients without WT1 mutations, patients with WT1 mutations had a worse 5-year event-free survival (21.7 ± 5.5% vs 48.9 ± 1.8%, P<0.001) and a worse overall survival (41.4 ± 6.6% vs 64.3 ± 1.7%, P<0.001). Moreover, patients with both WT1 and FLT3/ITD mutations had a dismal prognosis. Compared to chemotherapy alone, hematopoietic stem cell transplantation tended to improve the prognoses of WT1-mutated patients. Multivariate analysis demonstrated that WT1 mutations conferred an independent adverse impact on event-free survival (hazard ratio 1.910, P = 0.001) and overall survival (hazard ratio 1.709, P = 0.020). In conclusion, our findings have demonstrated that WT1 mutations are independent poor prognostic factors in pediatric AML.