WT1 Mutations Research Articles

Genetic mutational profiling analysis of T cell acute lymphoblastic leukemia reveal mutant FBXW7 as a prognostic indicator for inferior survival.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm for which there are currently no adequate biomarkers for developing risk-adapted therapeutic regimens to improve the treatment outcome. In this prospective study of 83 Chinese patients (54 children and 29 adults) with de novo T-ALL, we analyzed mutations in 11 T-ALL genes: NOTCH1, FBXW7, PHF6, PTEN, N-RAS, K-RAS, WT1, IL7R, PIK3CA, PIK3RA, and AKT1. NOTCH1 mutations were identified in 51.9 and 37.9% of pediatric and adult patients, respectively, and these patients showed improved overall survival (OS) and event-free survival (EFS). The FBXW7 mutant was present in 25.9 and 6.9% of pediatric and adult patients, respectively, and was associated with inferior OS and EFS in pediatric T-ALL. Multivariate analysis revealed that mutant FBXW7 was an independent prognostic indicator for inferior EFS (hazard ratio [HR] 4.38; 95% confidence interval [CI] 1.15-16.71; p = 0.03) and tended to be associated with reduced OS (HR 2.81; 95% CI 0.91-8.69; p = 0.074) in pediatric T-ALL. Mutant PHF6 was present in 13 and 20.7% of our childhood and adult cohorts, respectively, while PTEN mutations were noted in 11.1% of the pediatric patients. PTEN and NOTCH1 mutations were almost mutually exclusive, while IL7R and WT1 mutations were rare in pediatric T-ALL and PTPN11 and AKT1 mutations were infrequent in adult T-ALL. This study revealed differences in the mutational profiles of pediatric and adult T-ALL and suggests mutant FBXW7 as an independent prognostic indicator for inferior survival in pediatric T-ALL.

Variability in phenotype induced by the podocin variant R229Q plus a single pathogenic mutation.

BackgroundMutations in podocin (NPHS2) are the most common cause of childhood onset autosomal recessive steroid-resistant nephrotic syndrome (SRNS). The disease is characterized by early-onset proteinuria, resistance to immunosuppressive therapy and rapid progression to end-stage renal disease. Compound heterozygous changes involving the podocin variant R229Q combined with another pathogenic mutation have been associated with a mild phenotype with disease onset often in adulthood.MethodsWe screened 19 families with early-onset SRNS for mutations in NPHS2 and WT1 and identified four disease-causing mutations (three in NPHS2 and one in WT1) prior to planned whole-exome sequencing.ResultsWe describe two families with three individuals presenting in childhood who are compound heterozygous for R229Q and one other pathogenic NPHS2 mutation, either L327F or A297V. One child presented at age 4 years (A297V plus R229Q) and the other two at age 13 (L327F plus R229Q), one with steadily deteriorating renal function.ConclusionsThese cases highlight the phenotypic variability associated with the NPHS2 R229Q variant plus pathogenic mutation. Individuals may present with early aggressive disease.

Acute promyelocytic leukemia harbouring rare FLT3-TKD and WT1 mutations: A case report.

The involvement of the central nervous system (CNS) is rare in acute promyelocytic leukemia (APL). The present study reported the case of a 34-year-old male patient with APL that possessed a rare point mutation (p.Asn841Gly, c.2523C>A) in the tyrosine kinase domain of the FMS-like tyrosine kinase 3 (FLT3) gene and a novel Wilm tumor gene mutation (c.1209_1210insT/p.K404X). The patient suffered central nervous system and systemic relapses twice during systemic and intrathecal chemotherapy. At present, the patient is undergoing alternative induction and consolidation therapies, including the administration of FLT3 inhibitor, tetraarsenic tetrasulfide and novel cytotherapy, and is prepared for salvage allogeneic hematopoietic stem cell transplantion (allo-HSCT). The present study indicated that patients with APL that are at a high risk of relapse and unfavorable gene mutations should receive immediate allo-HSCT, whenever possible.

Epigenetic Aging Signatures Are Coherently Modified in Cancer.

Aging is associated with highly reproducible DNA methylation (DNAm) changes, which may contribute to higher prevalence of malignant diseases in the elderly. In this study, we analyzed epigenetic aging signatures in 5,621 DNAm profiles of 25 cancer types from The Cancer Genome Atlas (TCGA). Overall, age-associated DNAm patterns hardly reflect chronological age of cancer patients, but they are coherently modified in a non-stochastic manner, particularly at CpGs that become hypermethylated upon aging in non-malignant tissues. This coordinated regulation in epigenetic aging signatures can therefore be used for aberrant epigenetic age-predictions, which facilitate disease stratification. For example, in acute myeloid leukemia (AML) higher epigenetic age-predictions are associated with increased incidence of mutations in RUNX1, WT1, and IDH2, whereas mutations in TET2, TP53, and PML-PARA translocation are more frequent in younger age-predictions. Furthermore, epigenetic aging signatures correlate with overall survival in several types of cancer (such as lower grade glioma, glioblastoma multiforme, esophageal carcinoma, chromophobe renal cell carcinoma, cutaneous melanoma, lung squamous cell carcinoma, and neuroendocrine neoplasms). In conclusion, age-associated DNAm patterns in cancer are not related to chronological age of the patient, but they are coordinately regulated, particularly at CpGs that become hypermethylated in normal aging. Furthermore, the apparent epigenetic age-predictions correlate with clinical parameters and overall survival in several types of cancer, indicating that regulation of DNAm patterns in age-associated CpGs is relevant for cancer development.

Down-regulation of Wt1 activates Wnt/β-catenin signaling through modulating endocytic route of LRP6 in podocyte dysfunction in vitro

Podocyte dysfunction plays important roles in the pathogenesis of chronic kidney disease, and Wt1 has long been considered to be a marker of podocyte, whereas its roles and mechanisms in podocyte injury are still unclear though Wt1 mutations are reported to be involved in the development of glomerular disease in human and mice. Here we show that down-regulation of Wt1 could induce podocyte dysfunction and apoptosis through activating Wnt/β-catenin signaling. Podocytes treated with adriamycin demonstrated decreased expression of Wt1, coupled with activated Wnt/β-catenin signaling in vitro. Reduced expression of Wt1 in podocytes transfected with Wt1 siRNA is correlated with activated Wnt/β-catenin signaling, increased podocyte apoptosis, as well as suppressed expression of nephrin. Blockade of Wnt/β-catenin signaling with Dickkopf-1 ameliorated podocyte injury and apoptosis induced by Wt1 siRNA. We also found that membrane LRP6 was increased dramatically in podocytes transfected with Wt1 siRNA compared with control siRNA, while no significant change was found in total LRP6. Caveolin- and clathrin-dependent endocytosis were both involved in the regulation of β-catenin signaling. And we found that down-regulation of Wt1 in podocytes mediates activation of Wnt/β-catenin signaling by recruiting LRP6 to the caveolin-mediated endocytosis route, thereby sequestering it from clathrin-dependent endocytosis. As a result, we concluded that Wt1 expression levels in podocytes regulate Wnt/β-catenin signaling through modulating the endocytic fate of LRP6, and this indicates a potential target for the therapy of CKD.

Mutations in the BCR-ABL1 Kinase Domain and Elsewhere in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) has been the first human malignancy to be associated, more than 50 years ago, with a consistent chromosomal abnormality--the t(9;22)(q34;q11) chromosomal translocation. The resulting BCR-ABL1 fusion gene, encoding a tyrosine kinase with deregulated activity, has a central role in the pathogenesis of CML. Ancestral or additional genetic events necessary for CML to develop have long been hypothesized but never really demonstrated. CML can successfully be treated with tyrosine kinase inhibitors (TKIs). Mutations in the BCR-ABL1 kinase domain might arise, however, that confer resistance to 1 or more of the currently available TKIs. Hence, the critical role of BCR-ABL1 mutation screening for optimal therapeutic management, with the current gold standard technique, conventional sequencing, likely to be replaced soon by ultra-deep sequencing. Mutations in genes other than BCR-ABL1 include ASXL1, TET2, RUNX1, DNMT3A, EZH2, and TP53 in chronic phase patients and RUNX1, ASXL1, IKZF1, WT1, TET2, NPM1, IDH1, IDH2, NRAS, KRAS, CBL, TP53, CDKN2A, RB1, and GATA-2 mutations in advanced phase patients. The latter also display additional cytogenetic abnormalities, including submicroscopic regions of gain or loss that only single nucleotide polymorphism arrays or array comparative genomic hybridization can detect. Whether whole genome/exome sequencing studies will uncover novel mutations relevant for pathogenesis, progression, and risk-adapted therapy is still unclear.

Retrospective mutational analysis of NPHS1, NPHS2, WT1 and LAMB2 in children with steroid-resistant focal segmental glomerulosclerosis - a single-centre experience.

The aim of our study was to examine NPHS1, NPHS2, WT1 and LAMB2 mutations, previously reported in two thirds of patients with nephrotic syndrome with onset before the age of one year old. Genomic DNA samples from Polish children (n=33) with Steroid-Resistant Nephrotic Syndrome (SRNS) due to focal segmental glomerulosclerosis (FSGS), manifesting before the age of 13 years old, underwent retrospective analysis of NPHS1, NPHS2, WT1 (exons 8, 9 and adjacent exon/intron boundaries) and LAMB2. No pathogenic NPHS1 or LAMB2 mutations were found in our FSGS cohort. SRNS-causing mutations of NPHS2 and WT1 were detected in 7 of 33 patients (21%), including those with nephrotic syndrome manifesting before one year old: five of seven patients. Four patients had homozygous c.413G>A (p.Arg138Gln) NPHS2 mutations; one subject was homozygous for c.868G>A (p.Val290Met) NPHS2. A phenotypic female had C>T transition at position +4 of the WT1 intron 9 (c.1432+4C>T) splice-donor site, and another phenotypic female was heterozygous for G>A transition at position +5 (c.1432+5G>A). Genotyping revealed a female genotypic gender (46, XX) for the first subject and male (46, XY) for the latter. In addition, one patient was heterozygous for c.104dup (p.Arg36Profs*34) NPHS2; two patients carried a c.686G>A (p.Arg229Gln) NPHS2 non-neutral variant. Results indicate possible clustering of causative NPHS2 mutations in FSGS-proven SRNS with onset before age one year old, and provide additional evidence that patients with childhood steroid-resistant nephrotic syndrome due to focal segmental glomerulosclerosis should first undergo analysis of NPHS2 coding sequence and WT1 exons 8 and 9 and surrounding exon/intron boundary sequences, followed by gender genotyping.

ADCK4-Associated Glomerulopathy Causes Adolescence-Onset FSGS.

Hereditary defects of coenzyme Q10 biosynthesis cause steroid-resistant nephrotic syndrome (SRNS) as part of multiorgan involvement but may also contribute to isolated SRNS. Here, we report 26 patients from 12 families with recessive mutations in ADCK4. Mutation detection rate was 1.9% among 534 consecutively screened cases. Patients with ADCK4 mutations showed a largely renal-limited phenotype, with three subjects exhibiting occasional seizures, one subject exhibiting mild mental retardation, and one subject exhibiting retinitis pigmentosa. ADCK4 nephropathy presented during adolescence (median age, 14.1 years) with nephrotic-range proteinuria in 44% of patients and advanced CKD in 46% of patients at time of diagnosis. Renal biopsy specimens uniformly showed FSGS. Whereas 47% and 36% of patients with mutations in WT1 and NPHS2, respectively, progressed to ESRD before 10 years of age, ESRD occurred almost exclusively in the second decade of life in ADCK4 nephropathy. However, CKD progressed much faster during adolescence in ADCK4 than in WT1 and NPHS2 nephropathy, resulting in similar cumulative ESRD rates (>85% for each disorder) in the third decade of life. In conclusion, ADCK4-related glomerulopathy is an important novel differential diagnosis in adolescents with SRNS/FSGS and/or CKD of unknown origin.

46,XY disorder of sex development and wilms’ tumor due to mutation of WT1 gene: a case report

The Wilms' tumor suppressor gene (WT1) is a transcription factor that plays a major role in development of the gonads and kidneys. It is expressed even earlier than sex-determining region of the Y chromosome in the urogenital ridge from which the gonads and kidneys are derived. WT1 mutations will impair gonadal and urinary tract development and have been demonstrated to cause syndromes of WAGR, Denys–Drash and Fraiser. In this study, our aim is to identify mutation in WT1 gene and to describe clinical features of a Vietnamese patient with 46,XY disorder of sex development (DSD) associated with Wilms’ tumor. DNA was extracted from WBC and mutation analysis of WT1 gene was performed using PCR and direct sequencing. A 5 days newborn presented with penoscrotal hypospadias, microphallus, right testis in the right inguinal and no left testis was found. Karyotype was 46,XY and no ovaries and uterus were found using pelvic ultrasound. Wilms’ tumor was detected at 13 months of age by abdominal ultrasound and CT scan. Mutation analysis was identified a heterozygous missense mutation (c.1390G>A; p.D464N) in exon 9 of WT1 gene. In conclusions, WT1 analysis should be performed in newborns with complex hypospadias with at least one cryptorchid testis and in isolated 46,XY partial to complete gonadal dysgenesis. WT1 analysis is mandatory in all 46,XY DSD with associated kidney disease. Patients with WT1 mutations should be followed up closely because the risk of developing a Wilms' tumor, nephropathy. Written informed consent was obtained from the patient's parent or guardian for publication of this Case report (and any accompanying images). A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Incidences and Prognostic Impact of c-KIT, WT1, CEBPA, and CBL Mutations, and Mutations Associated With Epigenetic Modification in Core Binding Factor Acute Myeloid Leukemia: A Multicenter Study in a Korean Population.

BackgroundTo identify potential molecular prognostic markers in core binding factor (CBF) AML, we analyzed incidences and prognostic impacts of mutations in c-KIT, WT1, CEBPA, CBL, and a number of epigenetic genes in CBF AML.MethodsSeventy one and 21 AML patients with t(8;21) and inv(16) were enrolled in this study, respectively. NPM1, CEBPA, c-KIT, IDH1/2, DNMT3A, EZH2, WT1, and CBL mutations were analyzed by direct sequencing. Patients were categorized with respect to c-KIT and WT1 mutation status, and both clinical features and prognoses were compared.ResultsThe incidences of FLT3 internal tandem duplication (ITD), NPM1, CEBPA, IDH1/2, DNMT3A, EZH2, and CBL mutations were low (≤5%) in CBF AML patients. However, c-KIT and WT1 mutations occurred frequently (10.9% and 13.8%, respectively). t(8;21) patients with c-KIT mutations showed significantly shorter overall survival (OS) and disease free survival (DFS) periods than those without mutations (P<0.001, for both); however, although the limited number of t(8;21) patients were analyzed, WT1 mutation status did not affect prognosis significantly. Relapse or death during follow-up occurred more frequently in t(8;21) patients carrying c-KIT mutations than in those without the mutation, although the difference was significant only in a specific patient subgroup with no WT1 mutations (P=0.014).ConclusionsThe incidences of mutations in epigenetic genes are very low in CBF AML; however, c-KIT and WT1 mutations occur more frequently than others. The poor prognostic impact of c-KIT mutation in t(8;21) AML patients only applies in a specific patient subgroup without WT1 mutations. The prognostic impact of WT1 mutation in CBF AML is not evident and further investigation is required.

Clinical and molecular characterization of patients with heterozygous mutations in wilms tumor suppressor gene 1.

The Wilms tumor suppressor gene 1 (WT1) plays an essential role in urogenital and kidney development. Genotype/phenotype correlations of WT1 mutations with renal function and proteinuria have been observed in world-wide cohorts with nephrotic syndrome or Wilms tumor (WT). This study analyzed mid-European patients with known constitutional heterozygous mutations in WT1, including patients without proteinuria or WT. Retrospective analysis of genotype, phenotype, and treatment of 53 patients with WT1 mutation from all pediatric nephrology centers in Germany, Austria, and Switzerland performed from 2010 to 2012. Median age was 12.4 (interquartile range [IQR], 6-19) years. Forty-four of 53 (83%) patients had an exon mutation (36 missense, eight truncating), and nine of 53 (17%) had an intronic lysine-threonine-serine (KTS) splice site mutation. Fifty of 53 patients (94%) had proteinuria, which occurred at an earlier age in patients with missense mutations (0.6 [IQR, 0.1-1.5] years) than in those with truncating (9.7 [IQR, 5.7-11.9]; P<0.001) and splice site (4.0 [IQR, 2.6-6.6]; P=0.004) mutations. Thirteen of 50 (26%) were treated with steroids and remained irresponsive, while three of five partially responded to cyclosporine A. Seventy-three percent of all patients required RRT, those with missense mutations significantly earlier (at 1.1 [IQR, 0.01-9.3] years) than those with truncating mutations (16.5 [IQR, 16.5-16.8]; P<0.001) and splice site mutations (12.3 [IQR, 7.9-18.2]; P=0.002). Diffuse mesangial sclerosis was restricted to patients with missense mutations, while focal segmental sclerosis occurred in all groups. WT occurred only in patients with exon mutations (n=19). Fifty of 53 (94%) patients were karyotyped: Thirty-one (62%) had XY and 19 (38%) had XX chromosomes, and 96% of male karyotypes had urogenital malformations. Type and location of WT1 mutations have predictive value for the development of proteinuria, renal insufficiency, and WT. XY karyotype was more frequent and associated with urogenital malformations in most cases.

A high incidence of WT1 abnormality in bilateral Wilms tumours in Japan, and the penetrance rates in children with WT1 germline mutation.

Background:Bilateral Wilms tumours (BWTs) occur by germline mutation of various predisposing genes; one of which is WT1 whose abnormality was reported in 17–38% of BWTs in Caucasians, whereas no such studies have been conducted in East-Asians. Carriers with WT1 mutations are increasing because of improved survival.Methods:Statuses of WT1 and IGF2 were examined in 45 BWTs from 31 patients with WT1 sequencing and SNP array-based genomic analyses. The penetrance rates were estimated in WT1-mutant familial Wilms tumours collected from the present and previous studies.Results:We detected WT1 abnormalities in 25 (81%) of 31 patients and two families, which were included in the penetrance rate analysis of familial Wilms tumour. Of 35 BWTs from the 25 patients, 31 had small homozygous WT1 mutations and uniparental disomy of IGF2, while 4 had large 11p13 deletions with the retention of 11p heterozygosity. The penetrance rate was 100% if children inherited small WT1 mutations from their fathers, and 67% if inherited the mutations from their mothers, or inherited or had de novo 11p13 deletions irrespective of parental origin (P=0.057).Conclusions:The high incidence of WT1 abnormalities in Japanese BWTs sharply contrasts with the lower incidence in Caucasian counterparts, and the penetrance rates should be clarified for genetic counselling of survivors with WT1 mutations.

High frequency of cryptic chromosomal rearrangements involving the LMO2 gene in T-cell acute lymphoblastic leukemia

High frequency of cryptic chromosomal rearrangements involving the LMO2 gene in T-cell acute lymphoblastic leukemia

BCOR and BCORL1 mutations in pediatric acute myeloid leukemia

BCOR and BCORL1 mutations in pediatric acute myeloid leukemia

WT1 Recruits TET2 to Regulate Its Target Gene Expression and Suppress Leukemia Cell Proliferation

The TET2 DNA dioxygenase regulates cell identity and suppresses tumorigenesis by modulating DNA methylation and expression of a large number of genes. How TET2, like most other chromatin-modifying enzymes, is recruited to specific genomic sites is unknown. Here we report that WT1, a sequence-specific transcription factor, is mutated in a mutually exclusive manner with TET2, IDH1, and IDH2 in acute myeloid leukemia (AML). WT1 physically interacts with and recruits TET2 to its target genes to activate their expression. The interaction between WT1 and TET2 is disrupted by multiple AML-derived TET2 mutations. TET2 suppresses leukemia cell proliferation and colony formation in a manner dependent on WT1. These results provide a mechanism for targeting TET2 to a specific DNA sequence in the genome. Our results also provide an explanation for the mutual exclusivity of WT1 and TET2 mutations in AML, and suggest an IDH1/2-TET2-WT1 pathway in suppressing AML.

Integration of Cistromic and Transcriptomic Analyses Identifies Nphs2, Mafb, and Magi2 as Wilms' Tumor 1 Target Genes in Podocyte Differentiation and Maintenance.

The Wilms' tumor suppressor gene 1 (WT1) encodes a zinc finger transcription factor. Mutation of WT1 in humans leads to Wilms' tumor, a pediatric kidney tumor, or other kidney diseases, such as Denys-Drash and Frasier syndromes. We showed previously that inactivation of WT1 in podocytes of adult mice results in proteinuria, foot process effacement, and glomerulosclerosis. However, the WT1-dependent transcriptional network regulating podocyte development and maintenance in vivo remains unknown. Here, we performed chromatin immunoprecipitation followed by high-throughput sequencing with glomeruli from wild-type mice. Additionally, we performed a cDNA microarray screen on an inducible podocyte-specific WT1 knockout mouse model. By integration of cistromic and transcriptomic analyses, we identified the WT1 targetome in mature podocytes. To further analyze the function and targets of WT1 in podocyte maturation, we used an Nphs2-Cre model, in which WT1 is deleted during podocyte differentiation. These mice display anuria and kidney hemorrhage and die within 24 hours after birth. To address the evolutionary conservation of WT1 targets, we performed functional assays using zebrafish as a model and identified Nphs2, Mafb, and Magi2 as novel WT1 target genes required for podocyte development. Our data also show that both Mafb and Magi2 are required for normal development of the embryonic zebrafish kidney. Collectively, our work provides insights into the transcriptional networks controlled by WT1 and identifies novel WT1 target genes that mediate the function of WT1 in podocyte differentiation and maintenance.

DNA Hydroxymethylation Profiling Reveals That WT1 Mutations Result in Loss of TET2 Function in Acute Myeloid Leukemia

Mutations in TET2 and IDH1/2, which result in reduced conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), are observed in a significant proportion of patients with acute myeloid leukemia (AML). Hence these mutations define a novel class of AMLs with alterations in DNA methylation. However, it has been shown that not all AML cases with low levels of 5-hmC harbor somatic mutations in TET2 and IDH1/2. We hypothesized there are additional somatic mutations that can lead to alterations in TET enzyme function and resultant changes in the epigenetic state of hematopoietic cells.Mutational studies of the ECOG 1900 trial allowed us to discover that somatic mutations in the WT1 gene were mutually exclusive of IDH1/2 mutations and negatively correlated with TET2 mutations (Figure 1A). 28/313 (9%) of TET2/IDH-wild-type patients had somatic WT1 mutations, whereas 2/85 (2%) TET2/IDH1/2-mutant patients had co-occurring WT1 mutations (p=0.026, Fisher’s Exact test). Analysis of combined data from the ECOG 1900 study and the AML TCGA dataset confirmed a significant anti-correlation between WT1 mutations and TET2/IDH1/2 mutations (p=0.016). We next compared the DNA methylation profiles of WT1, TET2, and IDH1/2 mutant AML patients to AML patients wild-type for these disease alleles. Comparative analysis of the three hypermethylation profiles revealed a near-complete overlap of TET2 and WT1 hypermethylated loci within the IDH1/2 hypermethylation signature, and a highly significant overlap between the TET2 and WT1 mutant signatures (p-value< 0.001 for all comparisons), consistent with convergent, site-specific effects on DNA methylation.These genetic observations suggested a shared functional role for WT1, TET2, and IDH1/2 mutations in AML in regulating 5-hmC. WT1-mutant AML patients had significantly reduced 5-hmC (Liquid chromatography-electron spray ionization-tandem mass spectrometry) compared to AML patients wild-type for WT1, TET2 or IDH1/2 (p=0.016 T-test Figure 1B), similar to the reduction in 5-hmC observed in IDH1/2-mutant and TET2-mutant AML patient samples. In order to determine the impact of IDH1/2, WT1 and TET2 mutations on the distribution of epigenetic marks throughout the genome more precisely, we examined 5-hmC localization in primary AML specimens with next-generation sequencing. The average number of 5-hmC peaks present in samples from AML patients with TET2, WT1, IDH1 or IDH2 mutations was significantly reduced compared to AML patients wild-type for these disease alleles (t-test p-values between 0.0005 and 0.003). We observed highly significant overlap of differential 5-hmC peaks lost in WT1-mutant AML and TET2-mutant AML (Hypergeometric test p-value < 10-133). In a manner analogous to the findings for DNA methylation, the regions with reduced 5-hmC in WT1/TET2 mutant AML were a subset of those found in IDH1/2 mutant AML patients.We next investigated whether perturbations in WT1 expression can directly alter 5-hmC levels. Knockdown of Wt1 significantly decreased 5-hmC levels in M15 cells (p<0.01) (Figure 1C). Conversely, overexpression of WT1 in 32D cells significantly increased 5-hmC levels compared to cells expressing a control vector or WT1-mutant (p<0.05) (Figure 1D). Thus, WT1 can directly regulate 5-hmC levels. Given the effects of WT1 on 5-hmC levels and the inverse correlation between WT1 and TET2 mutations in AML, we hypothesized that WT1 might modulate TET2 function through direct interaction.Co-immunoprecipitation experiments demonstrated WT1 interacts with TET2 and with TET3. Consistent with these effects, WT1 expression inhibited colony forming ability of TET2-deficient cells, but could not attenuate the colony growth of TET2/TET3-deficient cells in vitro.Taken together, our genetic and functional studies demonstrate an inverse correlation between WT1 mutations and TET2/IDH1/2 mutations in AML and a functional role for WT1 in regulating DNA methylation. WT1 mutant AML is characterized by similar global and site-specific alterations in 5-hmC/5-mC as observed in TET2/IDH1/2 mutant AML. Furthermore, we demonstrate that alterations in WT1 levels directly regulate 5-hmC levels, which is due to an interaction between TET2/TET3 and WT1. These data provide a novel role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/2, and WT1 mutations define a novel AML subtype. [Display omitted] DisclosuresLevine:Novartis: Consultancy, Grant support Other.

Myeloid Cells Having NUP98-HOX Fusion Genes with Simultaneous FLT3, NRAS, or WT1 Mutations Confer Growth Advantage, Inhibit Differeniation/Apoptosis, and Augment Self-Renewal

Introduction: Clinical features and genetic backgrounds of acute myeloid Leukemia (AML) are heterogenous and the prognosis is poor. Cooperating alterations of several genes including oncogenes or tumor suppressor genes lead to development of Leukemia. AML leukemogenesis is thought to require at least two different types of genetic change: class I mutations, which confer a proliferative or survival advantage; and class II mutations, which inhibit myeloid differentiation and apoptotic activity. In hematological malignancies with 11p15 translocations, the NUP98 gene is reportedly fused to various partner genes, often including homeobox genes, such as HOXA9, A11, A13, C11, C13, D11, D13 and PMX. We previously revealed that high frequencies of FLT3 internal tandem duplication (FLT3-ITD), NRAS mutations (NRASMT), and WT1 mutations (WT1MT) in myeloid malignancies with NUP98-HOX fusions were found and their concurrent mutations with NUP98-HOX fusions were associated with clinical prognosis of the patients. However, biological outcome of the combination of NUP98-HOX fusions with FLT3-ITD, NRASMT or WT1MT remains to be investigated. To elucidate the mechanism resposible for the leukemogenesis, we examined the function of NUP98-HOX fusions and the concomitant somatic mutations in vitro. Methods: Full length of NUP98-HOXA9 (NHA9), FLT3-ITD (51 bp-duplication), NRASMT (G13V), and WT1MT (Arg250trp) was cloned into a plasmid that has a mammalian replication initiation region and a nuclear matrix attachment region, which possesses great ability to persist gene amplification for long periods. Each plasmid or the combination of the two plasmids (NHA9/FLT3-ITD, NHA9/NRASMT, NHA9/WT1MT) were transfected into IL3-dependent mouse myeloid 32D cell line. Cell proliferation was determined in medium without IL3. Cellular differentiation was examined in the medium containing G-CSF using May-Giemsa staining and fluorescence-activated cell sorting (FACS). Apoptosis was quantitated using FACS. Colony forming assay was performed in methyl cellulose medium with stem cell factor and IL3. Moreover, autophagy induced by tamoxifen was checked. Results: Compared to the empty vector, IL3-independent cellular growth was significantly increased in the cells transfected with NHA9 and FLT3-ITD alone, but not in NRASMT and WT1MT alone. In contrast, co-transfection of NRASMT or WT1MT with NHA9 significantly increased the IL3 independent proliferation of 32D cells. Similarly, cells transfected with NHA9/FLT3-ITD showed the enhanced IL3-independent proliferation. CD11b and Gr1, of which represent myeloid differentiation marker, decreased in all the transfected cells except for NRASMT. While transfection with FLT3-ITD, NRASMT and WT1MT increased the morphological nuclear segmentation of the cells, NHA9/FLT3-ITD, NHA9/NRASMT, and NHA9/WT1MT abrogated the nuclear segmentation. Both Annexin V positive/propidium iodide negative cells and active caspase 3-positive cells declined in all transfected cells. Transfection of NHA9/FLT3-ITD, NHA9/NRASMT, NHA9/WT1MT tended to increase the number of colony forming unit compared to the single gene-transfected cells. After treatment with tamoxifen, monodansylcadaverine, which was used to reveal autophagic vacuoles, was incorporated into empty vector-transfected cells, not into all transfected cells. Conclusions: NHA9 acquired proliferation capacity, blockage of differentiation, and anti-apoptosis activity, FLT3-ITD did proliferation capacity and anti-apoptosis activity, NRASMT and WT1MT did anti-apoptosis activity. By contrast, NHA9 with FLT3-ITD, NRASMT, or WT1MT conferred growth advantage, inhibited differentiation and apoptosis, augmented self-renewality. In vivo study demonstrated that NHA9 or each genetic mutation (FLT3-ITD and NRASMT) alone was insufficient to achieve myeloid leukemia. These suggested that leukemogenesis of NUP98-HOX fusion genes with simultaneous gene mutations might be induced by not only harmonization between class I and class II mutations but also self-renewality and anti-autophagic cell death. DisclosuresNo relevant conflicts of interest to declare.

Co-Existing Gene Mutations at Diagnosis and at Relapse in De Novo Acute Myeloid Leukemia with MLL Translocations

Background and purpose: The Mixed-lineage leukemia (MLL) gene located at 11q23 can be fused to a variety of partner genes through chromosomal translocations (MLL-T) in acute leukemia. The co-existence of commonly known mutated genes has not been comprehensively studied in MLL-T AML. We aimed to determine (1) the prevalence and clinical relevance of gene mutations at diagnosis, and (2) the genetic evolution profile at relapse in de novo AML with MLL-T.Materials and methods: Bone marrow samples from 80 de novo AML with MLL-T patients were analyzed on 20 gene mutations involving activating signaling pathway (class I), transcription pathway (class II), epigenetic regulators (class III), and tumor suppressors, TP53 and WT1 (class IV). MLL-T was all confirmed by FISH analysis. The common MLL fusion transcripts were detected by RT-PCR, including 26 MLLT3, 13 MLLT10, 12 MLLT4, 11 ELL, 3 MLLT1, 3 AFF1, and 2 EPS1. Another 6 infrequent or rare partner genes (one each of MLLT6,CBL, ARHGEF12, TET1, SEPT6, and SEPT9) were identified by cDNA panhandle PCR. In the remaining 4 cases, the partner genes were not identified. Mutational analyses were performed with PCR-based assays followed by direct sequencing. Twenty-two of 26 patients who relapsed had relapse samples for comparative analysis.Results: The frequencies of gene mutations of class I, II, III, and IV, in 80 de novo MLL-T AML patients were 49.4%, 3.8%, 10.3% and 1.3%, respectively. Together, 53.8% of patients with MLL-T had at least one mutated gene. KRAS (17.7%), FLT3-TKD (11.5%), and NRAS (11.4%) mutations occurred most frequently. Among the epigenetic regulators, 7.9% of patients had DNMT3A, 2.7% TET2, 1.4% ASXL1, and none had IDH1/2 or EZH2 mutations. Three patients had 3 co-existing mutations and 9 patients had two mutations, with 6 of the 12 carrying FLT3-TKD mutations. Gene mutations of transcription pathway and tumor suppressors rarely occurred, only involving NPM1 in two cases and one each for RUNX1 and WT1 mutation. The patients with DNMT3A mutations were significantly associated with older age (P=0.005), FAB AML-M4 (P<0.0001), and higher circulating monocytes (P=0.022). Patients with NRAS mutations had lower circulating monocytes (P<0.0001). KRAS-mutated patients had a higher percentage of marrow leukemic cells (P=0.002). No differences were observed between clinico-hematological features and mutation status of other genetic subtypes. Of the 65 MLL-T AML patients who received standard chemotherapy, 53 achieved complete remission. There were no differences in the outcomes between AML patients with MLLT3-MLL and MLL-MLLT10; patients with both subtypes had a superior event-free survival compared to those with other MLL-fusion transcripts (P=0.036) and a trend towards favorable overall survival (P=0.066). We failed to find significant differences between outcomes and the mutation status of each functional class. All the 3 patients carrying FLT3-ITD had an overall survival less than 6 months. Of the 22 paired diagnosis and relapse samples, 3 patients retained the same NRAS mutations at relapse whereas another one changed from G12D to G12C at relapse. Another 3 relapsed with identical KRAS mutations; while one acquired KRAS mutation at relapse. Two patients had stable DNMT3A mutations at both phases. All the 4 patients who harbored FLT3-TKD mutations at diagnosis lost the mutations at relapse. PTPN11 mutation was found in only one case at diagnosis, while none at relapse. One each had identical mutated gene of CBL and WT1 at both diagnosis and relapse. One each acquired TP53 and WT1 at relapse.Conclusions: Our results showed that co-existing mutations of RAS and FLT3-TKD were most frequently detected at diagnosis in de novo MLL-T AML. Gene mutations, except FLT3-ITD, had no impact on outcomes in MLL-T AML. FLT3-TKD mutations were absent in all relapse samples while clonal evolution of WT1, TP53 or KRAS mutations might contribute to the relapse of leukemia in a subset of MLL-T AML patients.Grant support: NHRI-EX93-9011SL, NHRI-EX96-9434SI, NSC95-2314-B-195-001, NSC96-2314-B-195-006-MY3, NSC97-2314-B-182-011-MY3, MMH-E-101-09 and OMRPG3C0021. DisclosuresNo relevant conflicts of interest to declare.

Mutation in Wilms’ Tumor 1 Induces DNA Hypermethylation of PRC2 Targets, Blocks Myelomonocytic Differentiation, and Defines a Novel Subtype of AML Responsive to EZH2 Inhibition

Wilms’ Tumor 1 is a transcription factor found to be recurrently mutated (WT1mut) in 10% of normal karyotype acute myeloid leukemia (NK-AML), predominantly in young adults with intermediate-poor prognosis, often in association with FLT3-ITD and high white cell count. Mutations are usually heterozygous and consist of small insertions or deletions clustered around exons 7 and 9, which encode zinc-finger DNA binding domains. In general, mutations disrupt these DNA binding domains leading to a truncated protein that may act in a dominant negative fashion. Currently, the mechanism by which WT1mut contributes to leukemogenesis is unknown, and no lead drug targets linked to this mutation have been identified to date. Using a novel computational method based on Boolean implications that link the presence of a somatic mutation to CpG methylation on a site-by-site basis, we found that mutation in WT1 is strongly linked to DNA hypermethylation in AML patient samples. In order to validate these findings, we expressed mutant WT1 protein prematurely truncated at exon 7 in THP-1 AML cells (confirmed to be wildtype for WT1 at both alleles), and after 10 passages we measured DNA methylation by 450K bead-chip arrays. We found consistent upregulation of DNA methylation in mutant but not wildtype WT1-expressing cells when compared to parental THP-1 cells, validating WT1 mutation as an active driver of DNA hypermethylation. Additional methylome analysis of human hematopoietic stem and progenitor compartments (HSPC) including (HSC, MPP, L-MPP, CMP, and GMP) indicated that WT1mut induces predominantly de novo DNA methylation, as virtually all CpG sites induced by the mutant protein are unmethylated in normal HSPC. Strikingly, the pattern of methylation in both WT1mut patient samples and WT1mut-THP-1 cells was enriched for polycomb repressor complex 2 (PRC2) target genes (p<1.6E-87), implicating a role for this repressive chromatin-remodelling complex in WT1mut leukemogenesis. In keeping with this, gene expression analysis of WT1mut AMLs (but not other normal karyotype AMLs) showed marked repression of known hematopoietic PRC2 target genes (as defined by Chip-Seq), suggesting WT1mut may induce a differentiation block through deregulation and hypermethylation of PRC2 targets.To explore this possibility, we expressed WT1mut in purified normal cord blood CD34+ HSPC using lentiviral transduction and performed in vitro liquid culture differentiation assays in IL-3, SCF, FLT3L, and GM-CSF. We found that WT1mut (but not wildtype or empty vector) induced a myelomonocytic differentiation block with fewer cells expressing CD11b, CD11c, and CD14. Separately, we also showed that WT1mut induced a differentiation block in a TF-1 cell model of erythroid differentiation. These findings suggest a role for WT1mut in perturbing myeloid differentiation in early HSPC.To test the therapeutic implications of our findings, we asked whether inhibition of the major enzymatic histone trimethylase component of PRC2, EZH2, could reverse the differentiation block caused by WT1mut. Significantly, we found that treatment of primary WT1mut AML blasts with the selective EZH2 inhibitor GSK-126 induced upregulation of the mature myeloid markers CD11b, CD33, and CD14. In contrast, NK-AML without WT1mut or acute promyleocytic leukemia cells did not show a significant differentiation response.Our results indicate that mutation in WT1 defines a novel subgroup of DNA hypermethyated AML with de novo hypermethylation of PRC2 target genes that may clinically respond to selective EZH2 inhibitors through differentiation. Importantly, our methods show that genome-wide analysis of mutation-specific DNA methylation patterns may have a future role in determining epigenetic therapies for personalized medicine. DisclosuresNo relevant conflicts of interest to declare.