Wilms Tumor 1 Mutations Research Articles

WT1 And DNMT3A Mutations in Prognostic Significance of Acute Myeloid Leukemia: A Meta-Analysis.

Background: Adult acute leukemia most commonly manifests as acute myeloid leukemia (AML), a highly heterogeneous malignant tumor of the blood system. The application of genetic diagnostic technology is currently prevalent in numerous clinical sectors. According to recent research, the presence of specific gene mutations or rearrangements in leukemia cells is the primary cause of the disease. As different types of leukemia are caused by atypical mutated genes, testing for these mutations or rearrangements can help diagnose leukemia and identify the disease's molecular targets for treatment. Methods: Using the search fields "WT1," "DNMT3A," "Acute myeloid leukemia," and "survival," the CBM, Cochrane Library, Scopus, EMBASE, and PUBMED databases were separately reviewed. The methodology for evaluating the risk of bias developed by the Cochrane Collaboration was used in conjunction with a methodical evaluation of pertinent literature. Excluded studies with the following characteristics: (1) incomplete and repetitive publications, (2) unable to retrieve or convert data, (3) non-English or Chinese articles. Results: This analysis included 13 studies covering a total of 3478 subjects. The frequency of Wilms' Tumor 1 (WT1) mutations is 6.7%-35.73%, and the frequency of DNMT3A mutations is 12.06%-51.1%. The remission rate of patients with WT1 mutations was less than that of patients without WT1 mutations (OR = 0.22; 95% confidence interval [CI]: 0.14, 0.36; p < 0.00001; I2 = 55%). The DNMT3A mutation has no statistical significance for the prognosis of AML (OR = 1.21; 95% CI: 0.93, 1.58; p = 0.16; I2 = 80%). After removing one study, the heterogeneity of the indicator (mitigation rate) among other studies of DNMT3A mutation was dramatically reduced (OR = 0.63; 95% CI: 0.43, 0.93; p = 0.02; I2 = 0%). Conclusions: Our meta-analysis shows that WT1 mutations hurt the remission rate of AML. Moreover, the impact of DNMT3A mutations on AML needs to be treated with caution. Gene diagnosis is critical for the prognosis and clinical management of AML.

WT1 gene mutations impact post-transplant relapse in myelodysplastic syndrome with excess blasts 2 patients.

Wilms tumor 1 (WT1) gene mutations are infrequent in myelodysplastic syndrome (MDS), but MDS with WT1 mutations (WT1mut) is considered high risk for acute myeloid leukemia (AML) transformation. The influence of WT1 mutations in patients with MDS after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is unclear. We performed a retrospective analysis of 136 MDS with excess blasts 2 (MDS-EB2) patients with available WT1 status who underwent their first allo-HSCT between 2017 and 2022 in our center. There were 20 (20/136, 15%) cases in the WT1mut group and 116 (116/136, 85%) cases in the WT1 wild-type (WT1wt) group. WT1mut patients had a higher 2-year cumulative incidence of relapse (CIR) than WT1wt cases (26.2% vs. 9.4%, p = 0.037) after allo-HSCT. Multivariate analysis of relapse showed that WT1 mutations (HR, 6.0; p = 0.002), TP53 mutations (HR, 4.2; p = 0.021), and ≥ 5% blasts in bone marrow (BM) at transplantation (HR, 6.6; p = 0.004) were independent risk factors for relapse. Patients were stratified into three groups according to the risk factors. Two-year CIR differed significantly in high-, intermediate-, and low-risk groups (31.8%, 11.6%, and 0%, respectively). Hence, WT1 mutations may be related to post-transplant relapse in patients with MDS-EB2, which warrants further study.

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

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

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

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

Prognostic Impact of WT1 Mutation on AML of Different Risk Groups Based on 2022 European Leukemianet (ELN) Risk Classification

Background The Wilms tumor 1 (WT1) gene, located on chromosome 11p13 exists in multiple isoforms. It has been implicated in the regulation of cell survival, proliferation and differentiation, and may function both as a tumor suppressor and an oncogene. Approximately 15% of acute myeloid leukemia (AML) patients carry WT1 mutations, and WT1 mutation have been shown to be independent predictor of worse clinical outcome in some but not all adult AML studies. Some studies suggested that NPM1-AML & AML with CEBPA double-mutation accompanied with WT1 mutation can alter their original favorable prognosis. Recently, a revised European LeukemiaNet (ELN) genetic risk classification has been published and the prognostic significance of WT1 mutation in three risk groups according to this 2022 ELIN risk classification is still unclear. Objective To investigate the prognostic significance of WT1 gene mutations in acute myeloid leukemia (AML) patients with different risk of the 2022-ELN risk classification. Methods 502 patients de novo AML diagnosed between March 2015 and July 2021 who underwent next-generation sequencing (NGS) tests and had MICM precise typing at the Peking University Institute of Hematology were enrolled in this study. The patients received chemotherapy ± hematopoietic stem cell transplantation (HSCT), and they were stratified into three groups according to the 2022 version of ELN-AML risk stratification. Results Among 502 patients, a total of 71 (14.1%) patients carried WT1 mutations, of which 57 patients had mutations at a single locus, 8 patients had mutations at double sites, 5 patients had co-mutations at 3 sites in the WT1 gene, and 1 patient had mutations at 4 sites. Comparing the incidence of AML-related mutations between the WT1 mutation group and the wild-type group, the results showed that the concomitant rate of NPM1 mutations was lower in the WT1 mutation group than in the WT1 wild-type group (14.1% vs 27.1%, p=0.028), and the concomitant rate of CEBPA-bZIP mutations was higher in the WT1 mutation group than in the WT1 wild-type group (29.6% vs 10.9%, p<0.001). The concomitant rate of CEBPA double mutation was also higher in the WT1 mutation group than in the WT1 wild-type group (29.6% vs 10.4%, p<0.001), and the concomitant rate of GATA2 mutation was higher in the WT1 mutation group than in the WT1 wild-type group (14.1% vs. 4.4%, p=0.004). In addition, the WT1 gene expression level (WT1/ABL1) was higher in the WT1 mutation group than in the WT1 wild group at diagnosis (22.0% vs 13.5%, p=0.004). All patients were further divided into 6 groups based on 2022 ELIN risk classification and whether they received HSCT. There were 158 (31.5%) patients in the low-risk chemotherapy group, 73 (14.5%) in the low-risk transplantation group, 72 (14.3%) in the intermediate-risk chemotherapy group, 58 (11.6%) in the intermediate-risk transplantation group, 78 (15.5%) in the high-risk chemotherapy group, and 63 (12.5%) in the high-risk transplantation group. Univariate and multivariate COX regression analyses were performed including WT1 mutation, FAB classification, age, sex, and karyotype stratification indicators. In 58 patients who were intermediate-risk and received HSCT, patients with WT1 mutation resulted in higher cumulative incidence of relapse(CIR) in compared with those without WT1 mutation (52.3% vs 33.5%, HR=9.28, p<0.001). In the remaining 5 groups of patients, WT1 mutation seemed have no significant effect on overall survival(OS), event-free survival (EFS), or CIR in patients (p-values were all less than 0.05). Conclusion Among intermediate-risk AML patients in the 2022 version of ELN stratification who received transplantation, the CIR after transplantation was higher in AML patients with WT1 mutation. It seemed that WT1 mutation was an independent risk factor for post-transplantation recurrence, and allogeneic HSCT cannot efficiently overcome the poor prognosis of WT1 mutation in the intermediate-risk group. Intermediate-risk AML with WT1 mutations still requires more active strategies to prevent relapse. Of course, the impact of WT1 mutation on prognosis needs to be further confirmed by expanding the sample size.

Downregulation of the Mutated WT1 Alleles in Malignant Hematopoietic Cells Via Nonsense-Mediated mRNA Decay Contradicts Their Prognostic Impact

Background The Wilms Tumor 1 (WT1) gene is highly expressed in almost all types of malignancies including acute myeloid leukemia (AML). It is also mutated in up to 20% of AML blasts in pediatric and adult patients. Both, the increased expression and also the presence of mutations are correlated negatively with chance of survival in different AML patient cohorts. We and others have previously showed devastating survival outcomes in children with AML blasts harboring WT1 mutations in combination FLT3-ITD and NUP98-NSD1 (Niktoreh et al., 2019). As WT1 can act as both, oncogene and tumor suppressor gene, this study was designed to analyze the WT1 genomic locus mutations in acute leukemia blasts. Methods and Results Screening for WT1 sequence alterations. Using the TruSight Myeloid Sequencing PanelTM (Illumina), we initially screened 35 leukemia and lymphoma cell lines for genomic alterations in 54 genes. This panel allowed us to detect even low variant-allele-frequency (VAF) alterations in the mutational hotspots of WT1, exons 7 and 9. In addition to a known WT1 mutation in CTS cells (AML M1), we discovered novel alterations in three other malignant cell lines. KASUMI-6 cells (AML M2) harbored the identical mutation as CTS cells, a 2-bp deletion in WT1 exon 7 leading to frameshift with a premature termination codon (c.1202_1203delGA, p.Arg401IlefsTer3). The U937 histiocytic lymphoma cells carried the known TP53 mutation and also an unknown heterozygous nonsense mutation in WT1 exon 7, changing the arginine 369 into a premature stop codon (c.1105C>T, p.Arg369Ter). Interestingly, MOLM-1 cells (CML in blast crisis) harbored a p.Arg462Gln alteration. Next, we performed Sanger sequencing for the complete coding region of WT1, including the upstream promoter as well as the 5'UTR and 3'UTR regions, all 10 exons and the adjacent 150 bp intronic sequences at the exon-intron boundaries. Sanger sequencing confirmed the mutations discovered via NGS and revealed single bp alterations in the WT1 promoter region of CTS cells and in the 5'UTR of K-562 cells. We also detected a recurring variation in intron 3 of WT1 (c.872+16G>A) in the cell lines K-562, NB-4, M07e and UOC-M1; the functional implications of the alterations are yet to be determined. Functional impact of WT1 mutations. To understand the potential impact of the WT1 sequence alterations, we studied the WT1 mRNA expression in these cells in the presence of cycloheximide (CHX), an inhibitor of the nonsense-mediated mRNA decay (NMD) machinery. As shown in figure 1A, U937 cells were used as negative control cells, as they are known to have no expression of the WT1 mRNA. Despite the presence of mutations in CTS, KASUMI-6 and MOLM-1 cells, all three cell lines expressed elevated levels of WT1, well within the range of 30 other malignant cell lines without any alterations/mutations in the WT1 locus (data not shown). To differentiate the WT1 expression between the mutated and the wildtype allele, we performed a fragment length analysis on a capillary electrophoresis system using the complimentary DNA of CHX-treated cell lines. For these analyses, we employed a multiplex PCR strategy using ABL1 as housekeeping gene and a primer pair binding in exons 4 and 10 of WT1. As delineated in figure 1B, the mutated alleles in CTS and KASUMI-6 cells, which are 2-bps shorter, could be easily separated from the wildtypes (data for KASUMI-6 not shown). Additionally, since our primer pair also amplified the alternatively spliced exon 5 and the alternative splice donor at the end of exon 9, leading to inclusion or exclusion of three amino acids (KTS), we were able to separately analyze the four major WT1 isoforms from both alleles. As shown in figure 1B, the expression levels of the WT1 mutated alleles of CHX-treated cells increased 2- to 3-fold compared to DMSO-treated cells in CTS and 3- to 4-fold in KASUMI-6 (data not shown). Conclusion Careful analyses of the WT1 genomic locus in 35 malignant hematological cell lines revealed previously unknown heterozygous loss-of-function WT1 mutations in KASUMI-6, MOLM-1 and U-937 cells. mRNA expression analysis demonstrated that these mutations are strongly regulated by NMD machinery, leading to (almost) complete downregulation of the mutated WT1 allele. These results strongly question any direct effect of WT1 mutations in the leukemic blasts and therefore cannot explain the role of mutations in WT1 as a prognostic factor in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A case of Potter sequence with WT1 mutation.

Wilms tumor 1 (WT1) is the causative gene of Denys-Drash syndrome and Frasier syndrome, and in most cases, kidney failure develops after birth. We report an unusual case of Potter sequence due to fetal nephropathy and kidney failure with a WT1 mutation. The neonate was born at 37weeks of gestation, and had no distinctive facial appearance or anomalies of the extremities. The external genitalia were ambiguous. Presence of a penile-like structure or hypertrophic clitoris was noted, and the urethra opened at the base of the penis or clitoris. On ultrasonographic examination, the kidney sizes were small. No kidney cysts were noted, but the kidney parenchymal luminosity was increased. Although the neonate received mechanical ventilation because of severe retractive breathing after birth, he died of poor oxygenation due to air leak syndrome at 60h after birth. The congenital anomalies of the kidney and urinary tract (CAKUT) gene panel revealed a heterozygous missense mutation in WT1 [NM_024426.6:exon9:c.1400G > A, p.(Arg467Gln)]. In WT1, missense mutations are associated with earlier onset of nephropathy than nonsense or splicing mutations. However, severe cases of fetal onset and early neonatal death with WT1 mutations are rare, and only one severe case with the same missense mutation in WT1 has been reported. Therefore, WT1 mutation may be suspected in Potter sequence patients with external genital abnormalities, and the WT1 missense mutation in our case [NM_024426.6:exon9:c.1400G > A, p.(Arg467Gln)] may indicate a severe case with fetal onset of nephropathy and kidney failure.

Frequency and clinical impact of WT1 mutations in the context of CEBPA-mutated acute myeloid leukemia

ABSTRACT Introduction Several studies have confirmed that mutations in the Wilms tumor 1 (WT1) gene occur in adult acute myeloid leukemia (AML). However, few data are available regarding the incidence of WT1 mutations in CEBPAmut AML and their impact. Methods We retrospectively analyzed the frequency and clinical impact of WT1 mutations in 220 newly diagnosed AML patients with CEBPA mutations(CEBPAmut). Chromosome karyotype analysis was performed by R or G banding method and further confirmed either by fluorescence in situ hybridization (FISH) and/or by multiple reverse transcription polymerase chain reaction (multiple RT–PCR). Mutations were detected with a panel of 112mutational genes using next-generation sequencing (NGS). Results Overall, 30 WT1 mutations were detected in 29 of the 220 CEBPAmut AML patients (13.18%) screened. These mutations clustered overwhelmingly in exon 7 (n=16). WT1 mutations were found to be significantly more frequent in AML patients with double-mutated CEBPA (CEBPAdm) than in AML patients with single-mutated CEBPA (17.36%vs. 8.08%, P = 0.043). Among WT1-mutated patients, the most common co-mutation was FLT3-ITD (n = 7, 24.14%), followed by NRAS (n = 5, 17.24%), CSF3R (n = 4, 13.79%), GATA2 (n = 4, 13.79%), and KIT (n = 4, 13.79%). The most frequent functional pathway was signaling pathways inas many as 62.07% of cases. Notably,the concomitant mutations in epigenetic regulatorswere inversely correlated with WT1 mutations(P = 0.003). CEBPAdm AML patients with WT1 mutations had inferior relapse-free survival, event-free survival and overall survival compared with patients CEBPAdm AML without WT1 mutations (P = 0.002, 0.004, and 0.010, respectively). Conclusion Our data showed that WT1 mutations are frequently identified in CEBPAmut AML, especially in CEBPAdm AML. CEBPAmut AML patients with WT1 mutations show distinct spectrum of comutations. In the context of CEBPAdm AML, WT1 mutations predict a poor prognosis.

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.

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.

Friend or foe? The case of Wilms' Tumor 1 (WT1) mutations in acute myeloid leukemia

Wilms tumor 1 (WT1) gene is commonly mutated in acute myeloid leukemia (AML), particularly in younger age population. The mechanism through which WT1 mutations drive leukemogenesis have not been fully elucidated; however, recent studies reported an association with the epigenetic pathway. Here, we studied the phenotypic characteristics and somatic mutational profile of 114 WT1-mutant AML patients and focused on potential WT1 gene relations to other cooperative genomic events that may impact disease prognosis. Invariant phenotypic and genomic associations of WT1 mutations in AML were uncovered and rigorously described. Our findings help improving the current understanding and definition of WT1-mutant AML patients' characteristics and clinical outcomes.

The Prognostic Impact of WT1 Expression Levels, Mutations, and SNP rs16754 in AML Patients: A Retrospective Cohort Study

Background & Objective: The clinical outcomes and treatment options for acute myeloid leukemia (AML) patients are highly dependent upon molecular markers. In this study, Wilms tumor 1 (WT1) (exons 7 and 9) mutations, single-nucleotide polymorphism (SNP) rs16754, and WT1 expression levels in 130 random AML patients were screened; FMs-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD), nucleophosmin (NPM1), and CCAAT/enhancer-binding protein alpha (CEBPA) mutations were also evaluated. Materials & Methods: Overall, 130 AML patients were recruited for this study. WT1 mutations were determined by Sanger sequencing, and expression levels were determined by real-time polymerase chain reaction (PCR). The Kaplan-Meier method was used to calculate overall survival (OS) and disease-free survival (DFS). Results: The frequency of WT1 mutations in the study population was 5.4, and it did not affect OS (P=0.98), DFS (P=0.97), or complete remission (CR) rates in AML patients. The major allele of SNP rs16754 in the current study was A. No significant differences were found for OS (P=0.52), DFS (P=0.42), or CR rates among all SNP rs16754 genotypes. The overexpression of WT1 was observed in 83 of patients at diagnosis. No significant difference was found for OS (P=0.84), DFS (P=0.82), or CR rates between AML patients with high and low WT1 expression levels. Conclusion: The results of the current study do not support WT1 mutation, SNP rs16754, or WT1 overexpression at diagnosis, as they were found to be poor prognostic markers in AML patients. © 2021, Zanjan University of Medical Sciences and Health Services. All rights reserved.

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.

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.

Analysis of pre-chemotherapy WBC, PLT, monocyte, hemoglobin, and MPV levels in acute myeloid leukemia patients with WT1, FLT3, or NPM gene mutations.

Effects of mutations on AML (acute myeloid leukemia) patients have been an area of clinical interest. The aim of this study was to analyze pre-chemotherapy WBC (white blood cell), platelet, monocyte, hemoglobin, and mean platelet volume (MPV) levels in acute myeloid leukemia patients with Wilms tumor 1 (WT1), FMS-like tyrosine kinase 3 (FLT3), or nucleophosmin (NPM) gene mutations, attempting to detect and compare possible differences in these values.The study included 71 patients with acute myeloid leukemia known to have WT1, FLT3, or NPM gene mutations. The patients were divided into 3 groups: FLT3-mutated AML patients without any accompanying known mutations other than WT1 at the time of diagnosis (Group 1), NPM-mutated AML patients without any accompanying known mutations other than WT1 at the time of diagnosis (Group 2), WT1-mutated AML patients with no other accompanying known mutations at the time of diagnosis (Group 3). We carried out intergroup comparisons of WBC, platelet (PLT), monocyte, hemoglobin, and MPV levels before chemotherapy.There was a statistically significant difference between the groups in terms of WBC parameters (P = .001). There were no statistically significant differences between the groups with respect to hemoglobin, platelet, and monocyte levels.Higher white blood cell counts could be observed in patients with FLT3-mutated AML.

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.

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.

Prognostic significance of The Wilms’ Tumor-1 (WT1) rs16754 polymorphism in acute myeloid leukemia

Acute myeloid leukemia is a genetically heterogeneous disease characterized by the accumulation of mutations in hematopoietic progenitor cells. For its heterogeneity, prognostic markers are very useful for therapeutic choice. The most important prognostic markers are age, white blood cell count, chromosomal alterations and gene mutations. Recent works have studied the prognostic significance of WT1 polymorphisms and mutations, highlighting the role of SNP rs16754 as a positive prognostic factor in AML patients. Nevertheless, the data are still unclear. To investigate the role of WT1 rs16754 polymorphism in AML, we designed a new tool for the detection using PNA directed PCR Clamping technology. Our data were able to establish a correlation between SNP rs16754 and the clinical outcome. Our results support the hypothesis that rs16754 polymorphism is an independent positive prognostic molecular marker that could be useful for therapeutic choice. In view of this, we described a novel assay faster, more sensitive and cheaper than DNA sequencing. The assay allows evaluating WT1 rs16754 polymorphism in diagnostic routine to improve prognostic information faster and without over-costing for diagnostic laboratories.

Description of mutation spectrum and polymorphism of Wilms' tumor 1 (WT1) gene in hypospadias patients in the Indonesian population

Hypospadias is one of the most common congenital anomalies of the penis. Previous studies reported mutation of the Wilms' tumor 1 (WT1) gene as a cause of hypospadias. The aim of this study is to describe the WT1 mutation spectrum and polymorphism in hypospadias patients in Indonesia. DNA was isolated from 74 hypospadias patients at the Division of Pediatric Surgery, Department of Surgery Hasan Sadikin Hospital. All exons in the WT1 gene were amplified by a PCR method, followed by Sanger sequencing. Mutation analysis was performed using BioEdit software and in silico analysis using Mutation Taster, Polymorphism Phenotyping-2 (PolyPhen-2), and Sorting Intolerant from Tolerant (SIFT). DNA analysis results showed two types of heterozygous mutations in five subjects (Table), hence the frequency of WT1 mutations was 6.7% (10/148 allele). The first mutation was a missense mutation identified in twin boys. The second was a novel heterozygous alteration in the non-coding region nine bp upstream of exon 6 (c.366-9T>C), which was identified in three patients. One heterozygous polymorphism in the coding region of exon 7 (c.471A>G/rs16754) was identified in 10 subjects. This variant did not cause any change in amino acid products (silence polymorphism). Allele frequency for the G allele (mutant allele) and A allele (wild type) was 13.5% and 86.5%, respectively. WT1 is one of the best known hypospadias genes. The WT1 gene is involved in male genital development in the early and late periods of sex determination, and hence is known as a long-term expression gene in genitalia development. Mutation analysis of WT1 in a Chinese population identified that the WT1 mutation frequency was 4.4%. The WT1 mutation frequency identified in the present study was higher, at 6.7%. Coincidentally, research subjects with p.R158H variants were monozygotic twin siblings with midshaft hypospadias accompanied by undescended testis in one and penoscrotal hypospadia with micropenis in the other. The incidence of familial hypospadias in male siblings suffering from hypospadias was reported to be 9.6% in a study conducted by Sorensen etal. Moreover, in the present study polymorphism c.471A>G(rs16754) at exon 7 was identified heterozygously in 10 research subjects (minor allele frequency 13.5%). WT1 mutations were identified in only a few cases of hypospadias and most of these were syndromic. This result implies that mutation of WT1 is not a common cause of hypospadias in the Indonesian population.

WT1 loss attenuates the TP53-induced DNA damage response in T-cell acute lymphoblastic leukemia.

Loss-of-function mutations and deletions in Wilms tumor 1 (WT1) gene are present in approximately 10% of T-cell acute lymphoblastic leukemia. Clinically, WT1 mutations are enriched in relapsed series and are associated to inferior relapse-free survival in thymic T-cell acute lymphoblastic leukemia cases. Here we demonstrate that WT1 plays a critical role in the response to DNA damage in T-cell leukemia. WT1 loss conferred resistance to DNA damaging agents and attenuated the transcriptional activation of important apoptotic regulators downstream of TP53 in TP53-competent MOLT4 T-leukemia cells but not in TP53-mutant T-cell acute lymphoblastic leukemia cell lines. Notably, WT1 loss positively affected the expression of the X-linked inhibitor of apoptosis protein, XIAP, and genetic or chemical inhibition with embelin (a XIAP inhibitor) significantly restored sensitivity to γ-radiation in both T-cell acute lymphoblastic leukemia cell lines and patient-derived xenografts. These results reveal an important role for the WT1 tumor suppressor gene in the response to DNA damage, and support the view that anti-XIAP targeted therapies could have a role in the treatment of WT1-mutant T-cell leukemia.